Modified variant bowman birk protease inhibitors

ABSTRACT

The present invention relates to modified variant Bowman Birk Protease Inhibitor proteins (BBPIs) that comprise peptides that bind target proteins, and that are further modified to have greater protease inhibitory activity and/or be produced at greater yields than the unmodified BBPIs. The invention encompasses polynucleotide constructs and expression vectors containing polynucleotide sequences that encode the modified variant BBPIs, the transformed host cells that express and produce the modified variant BBPIs, the modified variant BBPI proteins, the compositions comprising the modified variant BBPIs, and the methods for making and using the modified variant BBPIs in personal care.

1. FIELD OF THE INVENTION

The present invention relates to modified variant Bowman Birk Protease Inhibitor proteins (BBPIs) that comprise peptides that bind target proteins, and that are further modified to have greater protease inhibitory activity and/or be produced at greater yields than the unmodified BBPIs. The invention encompasses polynucleotide constructs and expression vectors containing polynucleotide sequences that encode the modified variant BBPIs, the transformed host cells that express and produce the modified variant BBPIs, the modified variant BBPI proteins, the compositions comprising the modified variant BBPIs, and the methods for making and using the modified variant BBPIs in personal care.

2. BACKGROUND OF THE INVENTION

Proteases are involved in a wide variety of biological processes. Disruption of the balance between proteases and protease inhibitors is often associated with pathologic tissue destruction.

Various studies have focused on the role of proteases in tissue injury, and it is thought that the balance between proteinases and proteinase inhibitors is a major determinant in maintaining tissue integrity. Serine proteinases from inflammatory cells, including neutrophils, are implicated in various inflammatory disorders, such as pulmonary emphysema, arthritis, atopic dermatitis and psoriasis. These and other inflammatory conditions are often associated with dysregulated levels of cytokines.

Proteases also degrade the vascular basement membrane and participate in the remodeling of the extracellular matrix to facilitate cell migration and invasion to promote tumor angiogenesis. Proteases release angiogenic growth factors bound to the extracellular matrix and to generate chemotactically active fragments derived from extracellular matrix components, which in turn exert chemotactic and mitogenic, modulatory activates on endothelial cells, smooth muscle cells and fibroblasts to participate in angiogenic processes. The list of protein factors angiogenically active in vivo includes fibroblast growth factors, Angiogenin, Angiopoietin-1, EGF, HGF, NPY, VEGF, TNF-alpha, TGF-beta, PD-ECGF, PDGF, IGF, IL8, and Growth hormone. Risks associated with current cytokine blocking agents include serious infections, anaphylaxis, and lupus-like syndrome. In addition, there is observed loss of clinical benefit after the drugs are stopped, and a small proportion of patients develop antibodies to the biological agents, which is likely to limit their efficacy with repeated use.

Synthetic and natural protease inhibitors have been shown to inhibit tumor promotion in vivo and in vitro. Previous research investigations have indicated that certain protease inhibitors belonging to a family of structurally-related proteins classified as serine protease inhibitors or SERPINS, are known to inhibit several proteases including trypsin, cathepsin G, thrombin, tissue kallikrein, as well as neutrophil elastase. The SERPINS are extremely effective at preventing/suppressing carcinogen-induced transformation in vitro and carcinogenesis in animal model systems. Systemic delivery of purified protease inhibitors reduces joint inflammation and cartilage and bone destruction as well.

Topical administration of protease inhibitors finds use in such conditions as atopic dermatitis, a common form of inflammation of the skin, which may be localized to a few patches or involve large portions of the body. The depigmenting activity of protease inhibitors and their capability to prevent ultraviolet-induced pigmentation have been demonstrated both in vitro and in vivo. Paine et al., Journal of Investigative Dermatology 116:587-595 [2001]. Also, protease inhibitors have been found to help wound healing (http://www.sciencedaily.com/releases/2000/10/001,002071718.htm). Secretory leukocyte protease inhibitor was demonstrated to reverse the tissue destruction and speed the wound healing process when applied topically. In addition, serine protease inhibitors can also help to reduce pain in lupus erythematosus patients (See U.S. Pat. No. 6,537,968).

Naturally occurring protease inhibitors can be found in a variety of foods such as cereal grains (oats, barley, and maize), Brussels sprouts, onion, beetroot, wheat, finger millet, and peanuts. One source of interest is the soybean. The average level in soybeans is around 1.4 percent and 0.6 percent for Kunitz and Bowman-Birk respectively, two of the most important protease inhibitors. These low levels make it impractical to isolate the natural protease inhibitor for clinical applications.

Thus, there is a need for a method to produce large quantities of protease inhibitors that have desired characteristics of protein therapeutics, and for compositions that effectively deliver the protease inhibitor in a usable form.

The compositions and methods according to the invention fulfill some of the above needs and in particular offer an advantage in providing protease inhibitors that specifically target the activity of cytokines involved in pathologic and non-pathologic processes.

3. SUMMARY OF THE INVENTION

The present invention relates to modified variant Bowman Birk Protease Inhibitor proteins (BBPIs) that comprise peptides that bind target proteins, and that are further modified to have greater protease inhibitory activity and/or be produced at greater yields than the unmodified BBPIs. The invention encompasses polynucleotide constructs and expression vectors containing polynucleotide sequences that encode the modified variant BBPIs, the transformed host cells that express and produce the modified variant BBPIs, the modified variant BBPI proteins, the compositions comprising the modified variant BBPIs, and the methods for making and using the modified variant BBPIs in personal care.

In one embodiment, the invention provides for an isolated modified variant Bowman Birk Protease Inhibitor (BBPI) that comprises a substituted amino acid at least at one amino acid position chosen from positions equivalent to 1, 4, 5, 11, 13, 18, 25, 27, 29, 31, 38, 40, 50, 52, 55, and 65 of the variant BBPI of SEQ ID NO:187, and in which the second protease inhibitory loop of the BBPI scaffold is a binding peptide. In one embodiment, the BBPI scaffold is chosen from the scaffolds: BBI (SEQ ID NO:13), BBIt (SEQ ID NO:185), BBI-AV (SEQ ID NO:186), BBIt-AV (SEQ ID NO:187), BBIt-VEGK (SEQ ID NO:640), BBIt-VEGT (SEQ ID NO:641), BBIt-VEGKD (SEQ ID NO:642), BBdb (SEQ ID NO:449), BBsb3 (SEQ ID NO:450), BBtc (SEQ ID NO:451), BBdb-AV (SEQ ID NO:452), BBsb3-AV (SEQ ID NO:453) and BBtc-AV (SEQ ID NO:454). In another embodiment, the binding peptide is chosen from a VEGF binding peptide, an FGF-5 binding peptide, a TGFβ binding peptide and a TNFα binding peptide. The VEGF-binding peptide in turn is chosen from ACYNLYGWTC (SEQ ID NO:9), KYYLYWW (SEQ ID NO:458), TLWKSYW (SEQ ID NO:459), DLYWW (SEQ ID NO:460), SKHSQIT (SEQ ID NO:468) KTNPSGS (SEQ ID NO:469) RPTGHSL (SEQ ID NO:470), KHSAKAE (SEQ ID NO:471) KPSSASS (SEQ ID NO:472), PVTKRVH (SEQ ID NO:473), TLHWWVT (SEQ ID NO:492), PYKASFY (SEQ ID NO:493), PLRTSHT (SEQ ID NO:494), EATPROT (SEQ ID NO:495), NPLHTLS (SEQ ID NO:496), KHERIWS (SEQ ID NO:497), ATNPPPM (SEQ ID NO:498), STTSPNM (SEQ ID NO:499), ADRSFRY (SEQ ID NO:500), PKADSKQ (SEQ ID NO:501), PNQSHLH (SEQ ID NO:502), SGSETWM (SEQ ID NO:503), ALSAPYS (SEQ ID NO:504), KMPTSKV (SEQ ID NO:505), ITPKRPY (SEQ ID NO:506), KWIVSET (SEQ ID NO:507), PNANAPS (SEQ ID NO:508), NVQSLPL (SEQ ID NO:509), TLWPTFW (SEQ ID NO:510), NLWPHFW (SEQ ID NO:511), SLWPAFW (SEQ ID NO:512), SLWPHFW (SEQ ID NO:513), APWNSHI (SEQ ID NO:514), APWNLHI (SEQ ID NO:515), LPSWHLR (SEQ ID NO:516), PTILEWY (SEQ ID NO:517), TLYPQFW (SEQ ID NO:518), HLAPSAV (SEQ ID NO:519), KYYLSWW (SEQ ID NO:520), WYTLYKW (SEQ ID NO:521), TYRLYWW (SEQ ID NO:522), RYSLYYW (SEQ ID NO:523), YYLYYWK (SEQ ID NO:524), NYQLYGW (SEQ ID NO:525), TKWPSYW (SEQ ID NO:226), TLWKSYW (SEQ ID NO:527), PLWPSYW (SEQ ID NO:528), RLWPSYW (SEQ ID NO:529), TLWPKYW (SEQ ID NO:530), KYDLYWW (SEQ ID NO;531), RYDLYWW (SEQ ID NO:532), DYRLYWW (SEQ ID NO:533), DYKLYWW (SEQ ID NO:534), EYKLYWW (SEQ ID NO:535), and RYPLYWW (SEQ ID NO:536). The FGF-5-binding peptide in turn is chosen from CACRTQPYPLCF (MM007; SEQ ID NO:430), CICTWIDSTPC (PS2; SEQ ID NO:431), CYGLPFTRC (SEQ ID NO:537), CEEIWTMLC (SEQ ID NO:538), CWALTVKTC (SEQ ID NO:539), CLTVLWTTC (SEQ ID NO:540), CTLWNRSPC (SEQ ID NO:541), CHYLLTNYC (SEQ ID NO:542), CRIHLAHKC (SEQ ID NO:543), TNIDSTP (SEQ ID NO:544), HLQTTET (SEQ ID NO:545), SLNNLTV (SEQ ID NO:546), TNIDSTP (SEQ ID NO:547), TNIDSTP (SEQ ID NO:548), LRILANK (SEQ ID NO:549), LLTPTLN (SEQ ID NO:550), ALPTHSN (SEQ ID NO:551), TNIDSTP (SEQ ID NO:552), LCRRFEN (SEQ ID NO:553), TNIDSTP (SEQ ID NO:554), TNIDSTP (SEQ ID NO:555), HLQTTET (SEQ ID NO:556), PLGLCPP (SEQ ID NO:557), GYFIPSI (SEQ ID NO:558), TKIDSTP (SEQ ID NO:559), HLQTTET (SEQ ID NO:560), WNIDSTP (SEQ ID NO:561), TWIDWTP (SEQ ID NO:562), RTQPYPL (SEQ ID NO:670) and TWIDSTP (SEQ ID NO:671). The TGFβ binding peptide in turn is chosen from CLCPENINVLPCN (PEN3; SEQ ID NO:436), CICKHNVDWLCF (MMO21W; SEQ ID NO:437), CICWTQHIHNCF (WTQ; SEQ ID NO:438), CVTTDWIEC (SEQ ID NO:563), CYYSQFHQC (SEQ ID NO:564), CPTLWTHMC (SEQ ID NO:565), QSACIVYYVGRKPKVECASSD (SEQ ID NO:566), QSACILYYIGKTPKIECASSD (SEQ ID NO:567), QSACILYYVGRTPKVECASSD (SEQ ID NO:568), acetyl-LCPENDNVSPCY-cohn2 (SEQ ID NO:569), KHNVRLL (SEQ ID NO:570), NDTPSYF (SEQ ID NO:571), AKLYAGS (SEQ ID NO:572), RGPAHSL (SEQ ID NO:573), NSLAERR (SEQ ID NO:574), HPLASPH (SEQ ID NO:575), QPWNKLK (SEQ ID NO:576), AWLr/Mipy (SEQ ID NO:577), PTKPAQQ (SEQ ID NO:578), PSLNRPQ (SEQ ID NO:579), HHARQEW (SEQ ID NO:580), RHHTPGP (SEQ ID NO:581), ASAINPH (SEQ ID NO:582), CHGYDRAPC (SEQ ID NO:644), CFAPADQAC (SEQ ID NO:645), CIPSRFITC (SEQ ID NO:646), CHGHTKLAC (SEQ ID NO:647), CNGKSKLAC (SEQ ID NO:648), PENINVLP (SEQ ID NO;672), KHNVDWL (SEQ ID NO:673) and WTQHIHNC (SEQ ID NO:674). The TNFα binding peptide in turn is chosen from RYWQDIP (T1; SEQ ID NO:474), APEPILA (T2; SEQ ID NO:475), DMIMVSI (T3; SEQ ID NO:476), WTPKPTQ (SEQ ID NO:583), ATFPNQS (SEQ ID NO:584), ASTVGGL (SEQ ID NO:585), TMLPYRP (SEQ ID NO:586), AWHSPSV (SEQ ID NO:587), TQSFSS (SEQ ID NO:588), THKNTLR (SEQ ID NO:589), GQTHFHV (SEQ ID NO:590), LPILTQT (SEQ ID NO:591), SILPVSH (SEQ ID NO:592), SQPIPI (SEQ ID NO:593), and QPLRKLP (SEQ ID NO:594).

In one embodiment, the invention provides for an isolated modified variant Bowman Birk Protease Inhibitor (BBPI) that comprises a substituted amino acid at least at one amino acid position chosen from positions equivalent to 1, 4, 5, 11, 13, 18, 25, 27, 29, 31, 38, 40, 50, 52, 55, and 65 of the variant BBPI of SEQ ID NO:187, and in which the second protease inhibitory loop of the BBPI scaffold is a binding peptide. In some embodiments, the substituted amino acid at position 1 chosen from 1A and 1C. In other embodiments, the substituted amino acid at position 4 is 4V. In other embodiments, the substituted amino acid at position 5 is chosen from P and A. In other embodiments, the substituted amino acid at position 13 is chosen from 13Y, 13I, 13F, 13M, 13L, 13V, 13K, and 13R. In other embodiments, the substituted amino acid at position 18 is chosen from 18I and 18V and 18L. In other embodiments, the substituted amino acid at position

25 is chosen from 25K, 25N, 25W, 25I, 25A and 25R. In other embodiments, the substituted amino acid at position 27 is chosen from 27H, 27K, 27V, 27A, and 27Q. In other embodiments, the substituted amino acid at position In other embodiments, the substituted amino acid at position 29 is chosen from 29R, 29K, and 29P. In other embodiments, the substituted amino acid at position 31 is chosen from 31Q, 31H, 31E, 31A, 31R, 31W, 31K and 31T. In other embodiments, the substituted amino acid at position 38 is chosen from 38N, 38K, and 38R. In other embodiments, the substituted amino acid at position 40 is chosen from 40H, 40K, 40Q, 40R, and 40Y. In other embodiments, the substituted amino acid at position 50 is chosen from 50R, 50Q, 50K, 50T, 50V, 50M, and 50S. In other embodiments, the substituted amino acid at position

In other embodiments, the substituted amino acid at position 52 is chosen from 52K, 52T, 52R, 52Q, 52L, 52H, 52A, 52M, 52S and 52E. In other embodiments, the substituted amino acid at position 55 is 55M. In other embodiments, the substituted amino acid at position 65 is chosen from 65E, 65Q, and 65D.

In other embodiments, the invention provides for an isolated modified variant Bowman Birk Protease Inhibitor (BBPI) that comprises an insert of SEQ ID NO: 389, a substituted amino acid at least at one amino acid position chosen from positions equivalent to 1, 4, 5, 11, 13, 18, 25, 27, 29, 31, 38, 40, 50, 52, 55, and 65 of the variant BBPI of SEQ ID NO:187, and in which the second protease inhibitory loop of the BBPI scaffold is a binding peptide.

In some embodiments, any one of the modified variant BBPIs described above is expressed as a fusion protein comprising the catalytic domain chosen from cellulase, cutinase and disulfide isomerase. In alternative embodiments, the modified variant BBPI is expressed as the fusion protein of SEQ ID NO:195.

In some embodiments, the modified variant BBPIs of the invention comprise a combination of at least two, at lest three, at least four, at least five, at least six, at least seven, and at least eight amino acid substitutions chosen from positions equivalent to 1, 4, 5, 11, 13, 18, 25, 27, 29, 31, 38, 40, 50, 52, 55, and 65 of the variant BBPI of SEQ ID NO:187. In some embodiments, the modified variant BBPI comprises a combination of at least two amino acid substitutions at amino acid positions equivalent to positions 50 and 52 of SEQ ID NO:187. An example of a modified variant BBPI comprising a combination of two amino acid substitutions at positions 50 and 52 is the modified variant BBPI of SEQ ID NO:595. In other embodiments, the modified variant BBPI comprises a combination of at least three amino acid substitutions at amino acid positions equivalent to positions 25, 29, 40, 50 and 52 of SEQ ID NO:187. In some embodiments, the combination of at least three amino acids is chosen from combinations 25L-50T-52A, 29P-50T-52A, 40K-50T-52A. Examples of modified variant BBPIs comprising a combination of three amino acid substitutions chosen from positions 25, 29, 40, 50 and 52 are the modified variant BBPI of SEQ ID NOS: 603, 607 and 609. In other embodiments, the modified variant BBPI comprises a combination of at least four amino acid substitutions chosen from amino acid substitutions at positions equivalent to positions 13, 29, 50 and 52 of SEQ ID NO:187. In some embodiments, the combination of at least four amino acids is chosen from combinations 13I-25L-50T-52A, 13I-29P-50T-52A, 13I-A0K-50T-52A, 25L-29P-50T-52A, 25L-40K-50T-52A, and 29P-40K-50T-52A. Examples of modified variant BBPIs comprising a combination of four amino acid substitutions chosen from positions 13, 29, 50 and 52 are the modified variant BBPIs of SEQ ID NOS:596, 600, 602, 604, 606, 608, and 643. In other embodiments, the modified variant BBPI comprises a combination of at least five amino acid substitutions chosen from amino acid substitutions at positions equivalent to positions 13, 25, 29, 40, 50, and 52 of SEQ ID NO:187. In some embodiments, the combination of at least five amino acids is chosen from combinations 13I-25L-29P-50T-52A, 13I-25L-40K-50T-52A, A13I-29P-40K-50T-52A, 25L-29P-40K-50T-52A, 13I-29P-40K-50K-52A, and 13I-29P-40K-50T-52T. Examples of modified variant BBPIs comprising a combination of five amino acid substitutions chosen from positions 13, 25, 29, 40, 50, and 52 are the modified variant BBPIs of SEQ ID NOS: 432, 434, 443, 445, 446, 597, 599, 601, 605, 615, 620, 624, and 625. In other embodiments, the modified variant BBPI comprises a combination of at least six amino acid substitutions chosen from amino acid substitutions at positions equivalent to positions 1, 4, 5, 11, 13, 25, 29, 40, 50 and 52 of SEQ ID NO:187. In some embodiments, the combination of at least six amino acids is chosen from combinations 13I-25L-29P-40K-50T-52A, 1C-13I-29P-40K-50T-52A, 4V-13I-29P-40K-50T-52A, 5P-13I-29P-40K-50T-52A, 11G-13I-29P-40K-50T-52A, 13I-25R-29P-40K-50T-52A, 13I-27R-29P-40K-50T-52A, 13I-29P-31A-40K-50T-52A, 13I-29P-31R-40K-50T-52A, 13I-29P-38N-40K-50T-52A, and 13I-29P-40K-50T-52A-65E. Examples of modified variant BBPIs comprising a combination of six amino acid substitutions chosen from positions 1, 4, 5, 11, 13, 25, 29, 40, 50 and 52 are the modified variant BBPIs of SEQ ID NOS: 598, 611, 612, 613, 614, 616, 619, 621, 622, 623, and 626.

In other embodiments, the modified variant BBPI comprises a combination of at least seven amino acid substitutions chosen from amino acid substitutions at positions equivalent to positions 13, 25, 29, 31, 40, 50 and 52 of SEQ ID NO:187. In some embodiments, the combination of at least seven amino acids is chosen from combinations 13L-25R-29P-31A-40K-50T-52A, 13L-25R-29P-31R-40K-50T-52A, and 13I-27A-29P-31A-50K-52T. Examples of modified variant BBPIs comprising a combination of seven amino acid substitutions chosen from positions 13, 25, 29, 31, 40, 50 and 52 are the modified variant BBPIs of SEQ ID NOS:491, 617, 618, and 632-639. In yet other embodiments, the modified variant BBPI comprises a combination of eight amino acid substitutions chosen from amino acid substitutions at positions equivalent to positions 13, 25, 27, 29, 31, 40, 50 and 52 of SEQ ID NO:187. In some embodiments, the combination of eight amino acids is chosen from combinations 13I-25R-27A-29P-31A-40H-50K-52T, 13I-25K-27A-29R-31E-40K-50Q-52Q, 13I-25K-27R-29E-31A-40H-50R-52K, 13I-25K-27A-29R-31A-40H-50R-52L and 13I-25K-27Q-29P-31E-40H-50R-52Q. Examples of modified variant BBPIs comprising a combination of eight amino acid substitutions chosen from positions 13, 25, 27, 29, 31, 40, 50 and 52 are the modified variant BBPIs of SEQ ID NOS: 627-631.

In some embodiments, the invention provides for an isolated modified variant Bowman Birk Protease Inhibitor (BBPI) that binds VEGF (VEGF-BBPI). The VEGF-BBPI comprises a substituted amino acid at least at one amino acid position chosen from positions equivalent to 1, 4, 5, 11, 13, 18, 25, 27, 29, 31, 38, 40, 50, 52, 55, and 65 of the variant BBPI of SEQ ID NO:187, and contains a VEGF-binding peptide that replaces the second protease inhibitory loop. In some embodiments, the VEGF-binding peptide is chosen from SEQ ID NOS:9, 458, 459, 460, 468, 469, 470, 471, 472 and 473. In some embodiments, the VEGF-BBPI has a polypeptide sequence chosen from SEQ ID NOS:601, 602, 627, 628, 630, 631, 643, 491, 632, 633, 634, 635, and 636.

In some embodiments, the isolated modified variant BBPI of the invention has greater trypsin inhibitory activity than the corresponding unmodified precursor variant BBPI. In other embodiments, the isolated modified variant BBPI of the invention has greater trypsin inhibitory activity and production yield than the corresponding unmodified precursor variant BBPI.

In some embodiments, the invention provides for an isolated modified variant Bowman Birk Protease Inhibitor (BBPI) of SEQ ID NO:187 is further comprises a substituted amino acid at least at one amino acid position chosen from positions equivalent to 13, 25, 27, 29, 31, 40, 50, and 52 of SEQ ID NO:187, and in which the VEGF-binding peptide is replaced by a variant peptide to bind a target protein chosen from FGF-5, TGFβ and TNFα.

In some embodiments, the VEGF-binding peptide comprised in the variant BBPI of SEQ ID NO:187 is replaced with an FGF-binding peptide having a sequence chosen from SEQ ID NOS: 430, 431, 670, and 671. In other embodiments, the VEGF-binding peptide is replaced with a TGFβ binding peptide chosen from SEQ ID NOS:436, 437, 438, 672, 673, and 674. In yet other embodiments, the VEGF-binding peptide is replaced with a TNFα binding peptide chosen from SEQ ID NOS:474, 475, and 476.

The invention also encompasses modified variant BBPIs that comprise VEGF-, FGF5-, TGFβ- or TNFα-binding peptides that have greater trypsin inhibitory activity and production yield than the corresponding unmodified precursor BBPI. In some embodiments, the modified variant BBPI comprising a FGF5-, TGFβ- or TNFα-binding peptide is chosen from the modified variant BBPIs of SEQ ID NOS:432, 434, 443, 445, 447, 637, 638, and 639.

In another embodiment, the invention provides for an isolated polynucleotide that encodes a BBPI fusion protein. The polynucleotide encoding the BBPI fusion protein comprises a first polynucleotide sequence that encodes the catalytic unit of an enzyme, and a second polynucleotide sequence encoding a modified variant BBPI e.g. a VEGF-binding BBPI (VEGF-BBPI), an FGF-binding BBPI (FGF-BBPI), a TGF-binding BBPI (TGF-BBPI) or a TNF-binding BBPI (TNF-BBPI). In some embodiments, the first polynucleotide sequence encodes the catalytic unit of a cellulase.

In another embodiment, the invention provides for a method for a modified variant BBPI in a bacterial cell, which comprises the step of mutating a polynucleotide that encodes a variant BBPI to generate a polynucleotide construct that encodes a modified variant BBPI comprising an amino acid substitution at least at one amino acid position chosen from positions equivalent to 1, 4, 5, 11, 13, 18, 25, 27, 29, 31, 38, 40, 50, 52, 55, and 65 of the variant BBPI of SEQ ID NO:187; the step of introducing the polynucleotide construct into a bacterial host cell; the step of culturing said bacterial cell under suitable culture conditions to allow expression of said heterologous DNA sequence; and the step of producing the modified variant BBPI, which has a greater trypsin inhibitory activity than the corresponding unmodified precursor variant BBPI. In some embodiments, the method of the invention further comprises the step of recovering the expressed modified variant BBPI. In other embodiments, the method of the invention also comprises activating the modified variant BBPI.

In some embodiments, the modified variant BBPI has a greater trypsin inhibitory activity and production yield than the corresponding unmodified precursor variant BBPI. Unmodified precursor variant BBPI are chosen from BBI-AV (SEQ ID NO:186), BBIt-AV (SEQ ID NO:187), BBdb-AV (SEQ ID NO:452), BBsb3 (SEQ ID NO:453), and BBtc-AV (SEQ ID NO:454), In some embodiments, the second protease inhibitory loop of the modified variant BBPI is a target protein binding peptide chosen from a VEGF-binding peptide, an FGF-5 binding peptide, a TGFβ binding peptide and a TNFα binding peptide.

Modified variant BBPIs that are produced by the method of the invention include but are not limited to modified variant BBPIs that comprise a combination of amino acid substitutions that are chosen from combinations of three substitutions e.g. 25L-50T-52A, 29P-50T-52A, 40K-50T-52A, four substitutions e.g. 13I-25L-50T-52A, 13I-29P-50T-52A, 13I-A0K-50T-52A, 25L-29P-50T-52A, 25L-40K-50T-52A, 29P-40K-50T-52A, five substitutions e.g. 13I-25L-29P-50T-52A, 13I-25L-40K-50T-52A, 25L-29P-40K-50T-52A,

13L-29P-40K-50T-52A, 13I-29P-40K-50K-52A, and 13L-29P-40K-50T-52T, six substitutions e.g. 13I-25L-29P-40K-50T-52A, 1C-13I-29P-40K-50T-52A, 4V-13I-29P-40K-50T-52A, 5P-13I-29P-40K-50T-52A, 11G-13I-29P-40K-50T-52A, 13I-25R-29P-40K-50T-52A, 13I-27R-29P-40K-50T-52A, 13I-29P-31A-40K-50T-52A, 13I-29P-31R-40K-50T-52A, 13I-29P-38N-40K-50T-52A, 13I-29P-40K-50T-52A-65E, seven substitutions e.g. 13L-25R-29P-31A-40K-50T-52A, 13L-25R-29P-31R-40K-50T-52A, 13I-25R-27A-29P-31A-50K-52T, and eight substitutions 13I-25R-27A-29P-31A-40H-50K-52T, 13I-25K-27A-29R-31E-40K-50Q-52Q, 13I-25K-27R-29E-31A-40H-50R-52K, 13I-25K-27A-29R-31A-40H-50R-52L, 13I-25K-27Q-29P-31E-40H-50R-52Q.

In another embodiment, the invention provides for an expression vector comprising a polynucleotide sequence that encodes a modified variant BBPI of the invention. As described above, the modified variant BBPI is a Bowman Birk Protease Inhibitor in which the second protease inhibitory loop has been replaced with a target protein binding peptide and which is further modified to contain an amino acid substitution at least at one amino acid position chosen from positions equivalent to 1, 4, 5, 11, 13, 18, 25, 27, 29, 31, 38, 40, 50, 52, 55, and 65 of the variant BBPI of SEQ ID NO:187. In some embodiments, the second protease inhibitory loop of the modified variant BBPI is a target protein binding peptide chosen from a VEGF-binding peptide, an FGF5-binding peptide, a TGFβ-binding peptide and a TNFα-binding peptide. In some embodiments, the modified BBPI is expressed as a protein having at least 80% identity to the polypeptide of SEQ ID NO:195.

In another embodiment, the invention provides for a host cell that is transformed with the vector of the invention, as described herein. In some embodiments, the host cell is a bacterial cell e.g. a Bacillus species host cell.

4. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-D provides the DNA and amino acid sequences of the aprE-BCE103-BBI-Histag expression cassette (EcoRI-HindIII) cloned into the pJM103 integration vector (SEQ ID NOS:1 and 2).

FIG. 2 provides a schematic map of the pJM103BBIhis expression vector.

FIG. 3 provides the DNA and amino acid sequences of 12BBIck81 from the BCE103 fusion site (at the BamHI) to the end of the gene (SEQ ID NOS:3 and 4). The CK37281 peptide sequence (ACYNLYGWTC (SEQ ID NO:9) is inserted into both the trypsin and chymotrypsin inhibitory loops.

FIG. 4 provides a graph showing titers of active versus inactive 2BBIck81 with various thiol reducing agents added at the given concentrations during the growth of the culture. The white portion of the bars represents the amount of active 2BBIck81 (determined by trypsin inhibition) and the black portion of the bars represents the amount of inactive 2BBIck81 (determined from BCE103 activity and assumes BCE103 and 2BBIck 81 are produced in 1:1 molar ratio). The fraction of active 2BBIck81 is indicated at the top of the bars. In this Figure, 37 C=no additions, BME=2-mercaptoethanol, Cys=cysteine, Glut=reduced glutathione, DTT=dithiothreitol.

FIG. 5 provides a graph showing the activation of 2BBIck81 with 2-mercaptoethanol (BME) after partial purification of the BCE-Ink2-2BBck81 fusion protein by ion exchange chromatography. The concentrations (μM) of BCE (black bars) and 2BBIck81 (white bars) measured by activity assays are shown before and after treatment with 3 mM 2-mercaptoethanol (BME).

FIG. 6 provides a graph showing results from a competition analysis of 2BBIck81 versus anti-VegF antibody for binding to VegF in a BioVeris assay. The binding competition with the CK37281 peptide is shown for comparison and the binding with the wild-type BBI is included as a negative control.

FIG. 7A-C provides the sequence of the synthetic DNA fragment carrying the H. insolens PDI-(hiPDI) that was inserted into the B. subtilis BBI expression vector (using the BssHII and SacI sites), as well as the amino acid sequence (SEQ ID NOS:5 and 6)

FIG. 8 A-C provides the DNA and amino acid sequences of the aprE-cutinase expression cassette that was ligated into the EcoRI-BamHI sites of p2JM103-Ink2-2BBIck81 (SEQ ID NOS:7 and 8).

FIG. 9 provides the amino acid sequence of the unmodified BBIt-AV protein (SEQ ID NO:187). Site-saturation libraries were constructed at all the numbered amino acids. The trypsin inhibitory loop and the VEGF binding peptide loop (in italics) are indicated and disulfide bonds are shown by the connecting lines. At the bottom, the sequence of the VEGF binding peptide is compared to the chymotrypsin inhibitory loop in sBBI that it replaces. The downward arrow indicates the potential protease cleavage site between the P₁ and P₁′ residues of the chymotrypsin reactive site.

FIG. 10 provides the relative BBI:BCE activity ratios for each amino acid substitution made at position 29. The ratios were averaged from quadruplicate cultures grown in microtiter plates in the absence (white bars) or presence (black bars) of 2-mercaptoethanol. The activity ratios for sBBI are included for comparison as a positive control. The ratios were normalized to the value determined for the wild-type amino acid in BBIt-AV (SEQ ID NO:187) (indicated by a horizontal line), leucine, that is shown in the absence (horizontally lined bar) or in the presence (diagonally lined bar) of 2-mercaptoethanol.

FIGS. 11A-B respectively provide the relative BBI:BCE activity ratios for the amino acid substitutions made at position 50 (A) and position 37 (B) (white bars). The ratios were averaged from quadruplicate cultures in the presence of 2-mercaptoethanol. The activity ratios for sBBI are included as a positive control for comparison (black bars). The ratios are normalized to the value (indicated by a horizontal line) determined for the wild-type amino acids in BBIt-AV (SEQ ID NO:187) and is shown with diagonally lined bars.

FIG. 12 provides the relative BBI:BCE activity ratios for the amino acid substitutions at the P₂′ position (residue 18 in FIG. 9, SEQ ID NO:187) in the trypsin inhibitory loop. The ratios were determined in quadruplicate in the presence of 2-mercaptoethanol (individual data points shown). The activity ratios for sBBI (BBI; SEQ ID NO:13), BBIt-AV (CT; SEQ ID N0187) and BBIt-AV-F50T (F50T) are shown on the right side of the horizontal line and were added for comparison. The ratios are normalized to the value (shown by a horizontal line) determined for BBIt-AV (CT).

FIG. 13 provides a comparison of the production of three BBIt-AVs containing combinations of 8 amino acid substitutions: octuple variant BBIt-AV-A13I-S25K-M27A-L29R-S31A-A40H-F50R-V52L (RL8; SEQ ID NO:630), BBIt-AV-A13I-S25K-M27A-L29R-S31E-A40K-F50Q-V52Q (QQ8; SEQ ID NO:628), and BBIt-AV-A13I-S25R-M27A-L29P-S31A-A40H-F50K-V52T (KT8; SEQ ID NO:627). The amount of active BBI species was determined by trypsin inhibition after growth (♦), after activation with 2-mercaptoethanol (▪) and after acid/heat treatment (▴). BBIt-AV, the quintuple variant BBIt-AV-A13I-L29P-A40K-F50T-V52A (TA5; SEQ ID NO:601) and sBBI are included for comparison.

FIG. 14 provides a comparison of the active BBI species (by trypsin inhibition) after growth (black bars), after activation with 2-mercaptoethanol (diagonally lined bars) and after acid/heat treatment (white bars) of different binding peptides (three TGFβ and two FGF5 binders) in the precursor variant BBPI scaffold (designated parent (PT)) versus the modified variant BBIt-AV-A13I-L29P-F50T-V52A scaffold (Q; SEQ ID NO:600). In all scaffolds, the binding peptides replaced the chymotrypsin inhibitory loop. PEN3 (SEQ ID NO:436), WTQ (SEQ ID NO:438) and MM021W (SEQ ID NO:437) are TGFβ binding peptides, and MM007 (SEQ ID NO:430) and FGFps2 (SEQ ID NO:431) are FGF5 binding peptides.

FIG. 15 provides the amino acid sequences of the variant Bowman Birk Inhibitor scaffolds from Dolichos biflorus (BBdb-AV, SEQ ID NO:452), the variant protease inhibitor IV or D-II (BBsb3-AV, SEQ ID NO:453) from Glycine max (soybean), and from Torresea (Amburana) cearensis (BBtc-AV, SEQ ID NO:454) in which the chymotrypsin loop is replaced with a VEGF-binding peptide, that are aligned to the sequence of the variant BBI, BBIt-AV (SEQ ID NO:187). The differences from the BBIt-AV sequence between C9 and C58 are shown underlined in bold font. In all scaffolds, the CK3781 binding peptide (ACYNLYGWT; SEQ ID NO:9) replaces the second protease inhibitory loop.

FIG. 16 provides a comparison of the active BBI concentration (by trypsin inhibition) of BBIt-AV, BBdb-AV and BBtc-AV after activation with 2-mercaptoethanol (white bars) and after acid/heat treatment (black bars).

FIG. 17 provides an alignment of unmodified variant BBIt-AV, (SEQ ID NO:187), BBdb-AV,(SEQ ID NO:452), BBsb3-AV (SEQ ID NO:453) and BBtc-AV (SEQ ID NO:454) and the corresponding unmodified precursor BBIt (SEQ ID NO:187), BBsb3 (SEQ ID NO:449), BBtc (SEQ ID NO:450) and BBdb (SEQ ID NO:451) BBPIs. The star symbol (*) identifies the invariant conserved C residues. The numbered residues identify the amino acids that are substituted to generate the modified variant BBPIs of the invention.

FIG. 18 Identification of T-cell epitopes in wild type BBI. (A) The stimulation indices (SI) values for all 71 donors were averaged and are shown, with standard deviations in error bars. (B) The percent responders to each peptide in the 20 wt BBI peptide set are shown.

5. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to modified variant Bowman Birk Protease Inhibitor proteins (BBPIs) that comprise peptides that bind target proteins, and that are modified to have greater protease inhibitory activity and/or be produced at greater yields than the unmodified BBPIs. The invention encompasses polynucleotide constructs and expression vectors containing polynucleotide sequences that encode the modified variant BBPIs, the transformed host cells that express and produce the modified variant BBPIs, the modified variant BBPI proteins, the compositions comprising the modified variant BBPIs, and the methods for making and using the modified variant BBPIs in personal care.

Unless otherwise indicated, the practice of the present invention involves conventional techniques commonly used in molecular biology, microbiology, protein purification, protein engineering, protein and DNA sequencing, and recombinant DNA fields, which are within the skill of the art. Such techniques are known to those of skill in the art and are described in numerous standard texts and reference works. All patents, patent applications, articles and publications mentioned herein are hereby expressly incorporated herein by reference in their entirety.

Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Various scientific dictionaries that include the terms included herein are well known and available to those in the art. Although any methods and materials similar or equivalent to those described herein find use in the practice or testing of the present invention, some preferred methods and materials are described. Accordingly, the terms defined immediately below are more fully described by reference to the Specification as a whole. It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary, depending upon the context they are used by those of skill in the art.

As used herein, the singular terms “a”, “an,” and “the” include the plural reference unless the context clearly indicates otherwise. Unless otherwise indicated, nucleic acids are written left to right in 5′ to 3′ orientation and amino acid sequences are written left to right in amino to carboxy orientation, respectively.

All patents, patent applications, and other publications, including all sequences disclosed within these references, referred to herein are expressly incorporated by reference, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference. All documents cited are, in relevant part, incorporated herein by reference. However, the citation of any document is not to be construed as an admission that it is prior art with respect to the present invention.

Numeric ranges are inclusive of the numbers defining the range. It is intended that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

The headings provided herein are not limitations of the various aspects or embodiments of the invention which can be had by reference to the Specification as a whole. Accordingly, as indicated above, the terms defined immediately below are more fully defined by reference to the specification as a whole.

5.1 DEFINITIONS

As used herein, the terms “isolated” and “purified” refer to a nucleic acid or amino acid (or other component) that is removed from at least one component with which it is naturally associated.

As used herein, the term “modified” when referring to a protease inhibitor (PI) e.g. Bowman Birk Protease Inhibitor (BBPI), refers to a BBPI having an amino acid sequence that is derived from the amino acid sequence of an unmodified “precursor” or “parent” BBPI protein. The unmodified precursor BBPI can be a naturally-occurring or wild-type protein, or a variant BBPI. The amino acid sequence of the modified protein is “derived” from the precursor protein amino acid sequence by the substitution, deletion or insertion of one or more amino acids of the region of the precursor amino acid sequence. In some embodiments, at least one amino acid is substituted to generate the modified protease inhibitor. In some embodiments, the parent protease inhibitor is a variant BBPI, which contains a trypsin and/or chotrypsin loop(s) that has been replaced with a variant sequence. Substitution of at least one amino acid of a variant precursor BBPI generates a modified variant BBPI protease inhibitor. In some embodiments, the modified variant PI comprises an amino acid substitution at least at one position equivalent to positions 1, 4, 5, 11, 13, 18, 25, 27, 29, 31, 38, 40, 50, 52, and 65 of SEQ ID NO:187 (DDESSKPCCDQCACTKSNPPQCRCSDMRLNSCHSACKSCACYNLYGWTCFCVDITDFCYEPCKPSE: SEQ ID NO:187). In other embodiments, the modified variant PI comprises a combination of substitutions as described herein. In yet other embodiments, the modified variant PI comprises an insertion. Such modifications are of the “precursor DNA sequence” which encodes the amino acid sequence of the precursor protease inhibitor rather than manipulation of the precursor protease inhibitor per se. The modified protease inhibitors herein encompass the substitution of any of the nineteen naturally occurring amino acids at any one of the amino acid residues in regions other than the reactive loops e.g. trypsin and/or chymotrypsin loops(s). The polynucleotides that encode the modified sequence are referred to as “modified polynucleotides”, and the polynucleotides that encode the precursor protease inhibitor are referred to as “precursor polynucleotides”.

As used herein, the term “protease inhibitor” (PI) herein refers to and is used interchangeably with Bowman Birk Protease Inhibitor (BBPI), which is a cysteine-rich protease inhibitor as described, for example, by Prakash et al. [J mol Evol 42:560-569 (1996)].

As used herein, the term “scaffold” refers to a BBPI protein sequence into which a variant sequence is introduced. In some embodiments, the scaffold is a variant scaffold, which has either the first protease inhibitory loop e.g. the trypsin loop, and/or the second protease inhibitory loop e.g. the chymotrypsin replaced with a binding peptide sequence. In other embodiments, the scaffold is a wild type BBPI scaffold The term “modified variant scaffold” refers to a variant scaffold that comprises modifications e.g. amino acid substitutions or insertions in the backbone of the BBPI scaffold.

As used herein, the term “backbone” when used in reference to a variant BBPI scaffold refers to the portion of the variant BBPI that is outside of the binding peptide sequence that has been introduced to replace the first and/or second protease inhibitory loop i.e. the trypsin and/or chymotrypsin loop. For example, the backbone of the variant BBPI of SEQ ID NO:187 refers to amino acids 1-40 and 50-66 i.e. the amino acids N-terminal and C-terminal to the invariant cysteine residues at amino acid positions 41 and 49, which bracket the VEGF binding sequence that was introduced to replace the chymotrypsin loop found in the wild type BBPI i.e. BBI of SEQ ID NO:13 (DDESSKPCCDQCACTKSNPPQCRCSDMRLNSCHSACKSCICALSYPAQCFCVDITDFCYEPCKPSEDDKEN; SEQ ID NO:13). In some embodiments, the scaffold has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the scaffold of the BBIt of SEQ ID NO:187.

As used herein, the terms “binding peptide”, “binding sequence”, “variant sequence” and “variant peptide” are used interchangeably, and refer to the short polypeptide sequence(s) that replace the first and/or second protease inhibitory loops of protease inhibitor e.g. Bowman Birk Inhibitor The binding peptide does not need to be of the same length as the protease inhibitory loop sequence it is replacing in the scaffold, and it differs from the protease inhibitory loop sequence of the wild-type BBPI by at least two amino acids. In some embodiments, replacing the first and/or second protease inhibitory loop e.g. trypsin and/or chymotrypsin loops of a precursor protease inhibitor alters the sequence that is equivalent to that spanning amino acids 15 to 21 (truypsin loop) and/or the sequence that is equivalent to that spanning amino acids 42 to 48 (chymotrypsin loop) of a wild-type e.g. SEQ ID NO:13 or variant SEQ ID NO:187 BBPI. The binding peptide sequence is heterologous to that of the protease inhibitor. Binding peptides bind to target proteins.

A “VEGF binding peptide”, an “FGF binding peptide”, a “TGF binding peptide” and a “TNF binding peptide” herein refer to a peptide sequence that binds VEGF, FGF5, TGFβ and TNFα, respectively.

A “VEGF composition”, an “FGF composition”, a “TGF composition” and a “TNF composition” herein refer to a composition e.g. a personal care composition, which comprises a VEGF-BBPI, an FGF-BBPI, a TGF-BBPI and a TGF-BBPI, respectively.

A “compound” when used in reference to a formulation described herein, refers to a modified variant BBPI e.g. a VEGF-BBPI, an FGF-BBPI, a TGF-BBPI or a TNF-BBPI. A formulation may include more than one type of modified variant BBPI i.e. the formulation may comprise a VEGF-BBPI and a TNF-BBPI, or any other combination of the four types of BBPIs disclosed herein.

The term “a” when used in reference to a modified variant BBPI comprised in a personal care composition, herein refers to a modified variant BBPI having a particular sequence. “a”, in this context does not limit the number of modified variant BBPI molecules that are needed in the personal care composition.

The terms “chymotrypsin loop” and “second protease inhibitory loop” are herein used interchangeably and refer to the sequence of amino acids that spans the amino acid sequence that corresponds to the second reactive site loop of a protease inhibitor of the Bowman Birk family. For example, the second protease inhibitory loop of the wild-type BBPI inhibitor from Glycine max i.e. BBI of SEQ ID NO:13 is the peptide sequence that spans the second reactive site loop encompassed by cysteine 10 and cysteine 11 (see Prakash et al. supra). C10 and C11 encompass an amino acid sequence that is equivalent to that which spans from amino acid 42 to amino acid 48 in the variant BBIt-AV of SEQ ID NO:187 (FIG. 9). The second protease inhibitory loop or chymotrypsin loop of a variant BBPI e.g. BBIt-AV of SEQ ID NO:187, corresponds to the amino acid sequence that spans amino acids 42-48 encompassed by the cysteines at positions 41 and 49, which are the cysteine residues equivalent to C10 and C11 as described by Prakash et al. While the amino acid sequence of second protease inhibitory loop of the wild-type BBI of SEQ ID NO:13 is a chymotrypsin inhibitory peptide, the second protease inhibitory loop of the variant BBIt-AV of SEQ ID NO:187 is a VEGF-binding peptide. Thus, the terms “chymotrypsin loop” or “second protease inhibitory loop” herein refer to the position of the loop and do not intend to imply that the sequence between C10 and C11 imparts protease inhibitory activity to a variant BBPI. Similarly, the terms “trypsin loop” and “first protease inhibitory loop” are herein used interchangeably and refer to the sequence of amino acids that spans the amino acid sequence that corresponds to the first reactive site loop of a protease inhibitor of the Bowman Birk family. For example, the second protease inhibitory loop of the wild-type BBPI inhibitor from Glycine max i.e. BBI of SEQ ID NO:13 is the peptide sequence that spans the second reactive site loop encompassed by cysteine 4 and cysteine 5 (see Prakash et al. supra). C4 and C5 encompass an amino acid sequence that is equivalent to that which spans from amino acid 15 to amino acid 21 in the variant BBIt-AV of SEQ ID NO:187 (FIG. 9).

As used herein, the term “target protein” refers to protein (e.g., enzyme, hormone, etc.), whose action would be blocked by the binding of the variant peptide. In some embodiments, the variant peptide binds the target protein when the peptide replaces the trypsin and/or chymotrypsin loop of a BBPI.

As used herein, “substituted” and “substitutions” refer to replacement(s) of an amino acid residue or nucleic acid base in a parent sequence. In some embodiments, the substitution involves the replacement of a naturally occurring residue or base. In some embodiments, two or more amino acids are substituted to generate a modified BBPI that comprises a combination of amino acid substitutions. In some embodiments, combinations of substitutions are denoted by the amino acid position at which the substitution is made. For example, a combination denoted by 25-50-52 means that three amino acids at positions 25, 50 and 52 are substituted. In other embodiments, the combination of substitutions is denoted by the amino acid position and the amino acid resulting from the substitution. For example, a modified BBPI that comprises the combination of substitutions 13I-25L-50T-52A is a modified BBPI wherein the amino acid at position 13 has been substituted with an isoleucine, the amino acid at position 25 has been substituted with a leucine, the amino acid at position 50 has been substituted with a threonine, and the amino acid at position 52 has been substituted with an alanine. Amino acid positions are positions equivalent to the numbered positions in the BBPI of SEQ ID NO:187. In some embodiments, the combination of substitutions is given in the context of the scaffold in which the substitutions are made. For example, the modified variant BBPI of SEQ ID NO:601 is also referred to as BBIt-AV-13I-29P-40K-50T-52A, indicating that the BBIt-AV scaffold (SEQ ID NO:187) has been modified to contain the resulting substitutions at amino acids 13, 29, 40, 50, and 52. In some embodiments, the original and substituted amino acid are indicated e.g. the modified variant BBPI of SEQ ID NO:601 can be referred to as BBIt-AV-A13I-L29P-A40K-F50T-V52A.

As used herein, “modification” and “modify” refer to any change(s) in an amino acid or nucleic acid sequence, including, but not limited to deletions, insertions, interruptions, and substitutions. In some embodiments, the modification involves the replacement of a naturally occurring residue or base. In other embodiments, the modification comprises a combination of at least one amino acid substitution. In yet other embodiments, the modification comprises an insertion with or without the insertion being combined with at least one amino acid substitution.

As used herein, the term “equivalent” when used in reference to an amino acid residue or the position of an amino acid residue in a BBPI refers to the position of an amino acid residue in a modified BBPI that corresponds in position in the primary sequence of the unmodified precursor BBPI. In order to establish the position of equivalent amino acid positions in a BBPI, the amino acid sequence of the BBPI that is modified is directly compared to the BBPI of SEQ ID NO:187, and in particular to the cysteine residues that are known to be invariant in protease inhibitors of the Bowman Birk Inhibitor family (Prakash et al. supra). After aligning the conserved cysteine residues, allowing for insertions and deletions in order to maintain alignment (i.e. avoiding the elimination of conserved cysteine residues through arbitrary deletion or insertion), the residues at positions equivalent to particular amino acid positions in the sequence of the BBPI of SEQ ID NO:187 are defined. Alignment of conserved cysteine residues preferably should conserve 100% of such residues. For example, in FIG. 17 the amino acid sequences of variant and wild-type BBPIs are aligned to provide the maximum amount of homology between amino acid sequences. A comparison of these sequences shows that the invariant cysteine residues are conserved. While the primary sequence is the preferred structure for determining the position of equivalent amino acids in the BBPIs of the invention, equivalent residues may also be identified by determining homology at the level of tertiary structure for a protein BBPI protein.

Equivalent amino acid positions are defined as those for which the atomic coordinates of two or more of the main chain atoms of a particular amino acid residue of the protein having putative equivalent residues and the protein of interest (N on N, CA on CA, C on C and O on O) are within 0.13 nm and preferably 0.1 nm after alignment. Alignment is achieved after the best model has been oriented and positioned to give the maximum overlap of atomic coordinates of non-hydrogen protein atoms of the proteins analyzed. The preferred model is the crystallographic model giving the lowest R factor for experimental diffraction data at the highest resolution available, determined using methods known to those skilled in the art of crystallography and protein characterization/analysis. The crystal structure of the Bowman Birk inhibitor from soybean has been determined (Hwang et al. J Biol. Chem. 10; 252(3):1099-101 [1977], Wei et al., J Biol. Chem. 10; 254(11):4892-4 [1979], Voss et al. Eur J Biochem. 15; 242(1):122-31 [1996]) and can be used as outlined above to determine equivalent amino acid positions on the level of tertiary structure.

As used herein, “fusion polypeptides,” “fusion proteins,” and “fusion analogs” encode from the amino-terminus a signal peptide functional as a secretory sequence functional in a host cell, a secreted polypeptide or portion thereof normally secreted from a host cell, a cleavable linker polypeptide and a desired polypeptide. In some embodiments, the fusion polypeptides include a spacer peptide positioned between the secretory sequence and a secreted polypeptide. In some embodiments, the fusion protein is processed by host cell enzymes (e.g., a protease), to yield the desired protein free from the other protein sequences in the fusion protein. As used herein, the terms “fusion analog,” “fusion polypeptide,” and “fusion protein” are used interchangeably.

As used herein, the term “activity” refers to any activity associated with a particular protein, such as enzymatic activity associated with a protease. In some embodiments, the activity is biological activity. In further embodiments, activity encompasses binding of proteins to receptors which results in measurable downstream effects (e.g., VEGF binding to its cognate receptor). “Biological activity” refers to any activity that would normally be attributed to that protein by one skilled in the art.

As used herein, “protease inhibitory activity” refers to the activity of a BBPI in inhibiting the proteolytic activity of a protease i.e. inhibiting the ability of a protease to hydrolyze peptides or substrates having peptide linkages.

As used herein, the term “expression” refers to the process by which a polypeptide is produced based on the nucleic acid sequence of a gene. The process includes both transcription and translation.

The term “production” with reference to a BBPI, encompasses the two processing steps of a full-length protease including: 1. the removal of the signal peptide, which is known to occur during protein secretion; and 2. the removal of the pro region, which creates the active mature form of the BBPI and which is known to occur during the maturation process (Wang et al., Biochemistry 37:3165-3171 (1998); Power et al., Proc Natl Acad Sci USA 83:3096-3100 (1986)).

As used herein, the term “production yield” refers to the level at which an unmodified and/or modified variant protease inhibitor e.g. a variant BBPI is produced. The greater the production yield, the greater the level or amount of protease inhibitor that is produced.

As used herein, the term “efficient production” herein to the production of a protein e.g. a modified variant BBPI, and implies that said protein is produced at a level that is greater than that of an unmodified or precursor variant BBPI.

As used herein, the term “substantially pure” when applied to the proteins or fragments thereof of the present invention means that the proteins are essentially free of other substances to an extent practical and appropriate for their intended use. In particular, the proteins are sufficiently pure and are sufficiently free from other biological constituents of the host cells so as to be useful in, for example, protein sequencing, and/or producing pharmaceutical preparations.

As used herein, the term “substantially free” encompasses preparations of the desired polypeptide having less than about 20% (by dry weight) other proteins (i.e., contaminating protein), less than about 10% other proteins, less than about 5% other proteins, or less than about 1% other proteins.

As used herein, the terms “polynucleotide”, “nucleic acid molecule” and “nucleic acid sequence” include sequences of any form of nucleic acid, including, but not limited to RNA, DNA and cDNA molecules. It will be understood that, as a result of the degeneracy of the genetic code, a multitude of nucleotide sequences encoding a given protein may be produced.

As used herein, the terms “DNA construct”, “polynucleotide construct” and “transforming DNA” are used interchangeably to refer to DNA used to introduce sequences into a host cell or organism. The DNA may be generated in vitro by PCR or any other suitable technique(s) known to those in the art. In some embodiments, the DNA construct comprises a sequence of interest (e.g., a modified sequence). In some embodiments, the sequence is operably linked to additional elements such as control elements (e.g., promoters, etc.). In some embodiments, the DNA construct comprises sequences homologous to the host cell chromosome. In other embodiments, the DNA construct comprises non-homologous sequences. Once the DNA construct is assembled in vitro it may be used to mutagenize a region of the host cell chromosome (i.e., replace an endogenous sequence with a heterologous sequence).

As used herein, the term “heterologous DNA sequence” refers to a DNA sequence that does not naturally occur in a host cell. In some embodiments, a heterologous DNA sequence is a chimeric DNA sequence that is comprised of parts of different genes, including regulatory elements.

As used herein, the term “heterologous protein” refers to a protein or polypeptide that does not naturally occur in the host cell i.e. it is encoded by a heterologous sequence.

As used herein, “homologous protein” refers to a protein or polypeptide native or naturally occurring in a cell.

As used herein, the term “vector” refers to a polynucleotide construct designed to introduce nucleic acids into one or more cell types. Vectors include cloning vectors, expression vectors, shuttle vectors, and plasmids. In some embodiments, the polynucleotide construct comprises a DNA sequence encoding a modified variant BBPI.

As used herein, the term “expression vector” refers to a vector that has the ability to incorporate and express heterologous DNA fragments in a foreign cell. Many prokaryotic and eukaryotic expression vectors are commercially available. Selection of appropriate expression vectors is within the knowledge of those of skill in the art.

As used herein, the term “plasmid” refers to a circular double-stranded (ds) DNA construct used as a cloning vector, and which forms an extrachromosomal self-replicating genetic element in some eukaryotes or prokaryotes, or integrates into the host chromosome.

As used herein in the context of introducing a nucleic acid sequence into a cell, the term “introduced” refers to any method suitable for transferring the nucleic acid sequence into the cell. Such methods for introduction include but are not limited to protoplast fusion, transfection, transformation, conjugation, and transduction (See e.g., Ferrari et al., “Genetics,” in Hardwood et al, (eds.), Bacillus, Plenum Publishing Corp., pages 57-72, [1989]).

As used herein, the terms “transformed” and “stably transformed” refers to a cell that has a non-native (heterologous) polynucleotide sequence integrated into its genome or as an episomal plasmid that is maintained for at least two generations.

As used herein, “percent (%) sequence identity” or “percent homology” when used in reference to a polynucleotide or to a polypeptide sequence is defined as the percentage of nucleotide or amino acid residues in a candidate sequence that are identical with the nucleotide or amino acid residues of a sequence disclosed herein. The percent identity shared by polynucleotide or polypeptide sequences is determined by direct comparison of the sequence information between the molecules by aligning the sequences and determining the identity by methods known in the art. In some embodiments, the alignment includes the introduction of gaps in the sequences to be aligned. In addition, for sequences which contain either more or fewer nucleotides or amino acids than those of the candidate polynucleotide or polypeptide sequences, it is understood that the percentage of homology will be determined based on the number of homologous nucleotides or amino acids in relation to the total number of nucleotides or amino acids. Thus, for example, homology of sequences shorter than those of the sequences identified herein will be determined using the number of nucleosites or amino acids in the shorter sequence. This homology is determined using standard techniques known in the art (See e.g., Smith and Waterman, Adv. Appl. Math., 2:482 [1981]; Needleman and Wunsch, J. Mol. Biol., 48:443 [1970]; Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444 [1988]; programs such as GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package (Genetics Computer Group, Madison, Wis.); and Devereux et al., Nucl. Acid Res., 12:387-395 [1984]).

As used herein, an “analogous sequence” is one wherein the function of the protein is essentially the same as that designated for the Bowman Birk family of protease inhibitors. Additionally, analogous proteins include at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% sequence identity with the sequence of the variant BBPI of SEQ ID NO:187. Analogous sequences are determined by known methods of sequence alignment. A commonly used alignment method is BLAST, although as indicated above and below, there are other methods that also find use in aligning sequences.

One example of a useful algorithm is PILEUP. PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments. It can also plot a tree showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng and Doolittle (Feng and Doolittle, J. Mol. Evol., 35:351-360 [1987]). The method is similar to that described by Higgins and Sharp (Higgins and Sharp, CABIOS 5:151-153 [1989]). Useful PILEUP parameters including a default gap weight of 3.00, a default gap length weight of 0.10, and weighted end gaps.

Another example of a useful algorithm is the BLAST algorithm, described by Altschul et al., (Altschul et al., J. Mol. Biol., 215:403-410, [1990]; and Karlin et al., Proc. Natl. Acad. Sci. USA 90:5873-5787 [1993]). A particularly useful BLAST program is the WU-BLAST-2 program (See, Altschul et al., Meth. Enzymol., 266:460-480 [1996]). WU-BLAST-2 uses several search parameters, most of which are set to the default values. The adjustable parameters are set with the following values: overlap span=1, overlap fraction=0.125, word threshold (T)=11. The HSP S and HSP S2 parameters are dynamic values and are established by the program itself depending upon the composition of the particular sequence and composition of the particular database against which the sequence of interest is being searched. However, the values may be adjusted to increase sensitivity. A % amino acid sequence identity value is determined by the number of matching identical residues divided by the total number of residues of the “longer” sequence in the aligned region. The “longer” sequence is the one having the most actual residues in the aligned region (gaps introduced by WU-Blast-2 to maximize the alignment score are ignored). A preferred method utilizes the BLASTN module of WU-BLAST-2 set to the default parameters, with overlap span and overlap fraction set to 1 and 0.125, respectively.

A “host cell” refers to a suitable cell from a cell that serves as a host for an expression vector comprising DNA according to the present invention. A suitable host cell may be a naturally occurring or wild-type host cell, or it may be an altered host cell. In one embodiment, the host cell is a Gram positive microorganism. In some preferred embodiments, the term refers to cells in the genus Bacillus.

As used herein, “Bacillus sp.” includes all species within the genus “Bacillus,” as known to those of skill in the art, including but not limited to B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. clausii, B. halodurans, B. megaterium, B. coagulans, B. circulans, B. lautus, and B. thuringiensis. It is recognized that the genus Bacillus continues to undergo taxonomical reorganization. Thus, it is intended that the genus include species that have been reclassified, including but not limited to such organisms as B. stearothermophilus, which is now named “Geobacillus stearothermophilus.” The production of resistant endospores in the presence of oxygen is considered the defining feature of the genus Bacillus, although this characteristic also applies to the recently named Alicyclobacillus, Amphibacillus, Aneurinibacillus, Anoxybacillus, Brevibacillus, Filobacillus, Gracilibacillus, Halobacillus, Paenibacillus, Salibacillus, Thermobacillus, Ureibacillus, and Virgibacillus.

As used herein, a “promoter sequence” refers to a DNA sequence which is recognized by the bacterial host for expression purposes. In preferred embodiments, it is operably linked to a DNA sequence encoding the fusion polypeptide. Such linkage comprises positioning of the promoter with respect to the translation initiation codon of the DNA sequence encoding the fusion DNA sequence. In particularly preferred embodiments, the promoter sequence contains transcription and translation control sequences which mediate the expression of the fusion DNA sequence.

As used herein, a nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA encoding a secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Operably linked DNA sequences are usually contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

As used herein, “recombinant” includes reference to a cell or vector, that has been modified by the introduction of a heterologous nucleic acid sequence or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found in identical form within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all as a result of deliberate human intervention.

As used herein, the term “personal care composition” refers to a product for application to the skin, hair, nails, oral cavity and related membranes for the purposes of improving, cleaning, beautifying, treating, and/or caring for these surfaces and membranes. In some embodiments, the personal care composition is in the form of an emulsified vehicle, such as a nutrient cream or lotion, a stabilized gel or dispersioning system, such as skin softener, a nutrient emulsion, a nutrient cream, a massage cream, a treatment serum, a liposomal delivery system, a topical facial pack or mask, a surfactant-based cleansing system such as a shampoo or body wash, an aerosolized or sprayed dispersion or emulsion, a hair or skin conditioner, styling aid, or a pigmented product such as makeup in liquid, cream, solid, anhydrous or pencil form. However, it is not intended that the present invention be limited to any particular form, as various forms find use in the present invention.

Personal care products can be classified/described as cosmetic, over-the-counter (“OTC”) compounds that find use in personal care applications (e.g., cosmetics, skin care, oral care, hair care, nail care). In some embodiments, the modified variant BBPI is added to a personal care composition such as a hair care composition, a skin care composition, a nail care composition, a cosmetic composition, or any combinations thereof.

As used herein, “skin care composition” refers to compositions that are applied to skin in order to provide beneficial properties, including but not limited to wrinkle minimizing, wrinkle removal, decoloring, coloring, skin softening, skin smoothing, depilation, cleansing, etc. In some particularly preferred embodiments, the present invention provides skin care compositions that improve skin tone. In these embodiments, the improvement comprises lessening of wrinkles, smoothing skin texture, modifying skin coloration, and other desired cosmetic benefits. In further embodiments, the skin care composition is in a form selected from the group consisting of body washes, moisturizing body washes, deodorant body washes, antimicrobial cleansers, skin protecting treatments, body lotions, facial creams, moisturizing creams, facial cleansing emulsions, surfactant-based facial cleansers, facial exfoliating gels, facial toners, exfoliating creams, facial masks, after shave lotions, balms, and/or radioprotective compositions (e.g., sunscreens).

As used herein, “cosmetic composition” refers to compositions that find use in the cosmetics. The Food Drug and Cosmetic Act (FD&C Act) definition is used herein. Thus, cosmetics are defined by their intended use, as articles intended to be rubbed, poured, sprinkled, or sprayed on, introduced into, or otherwise applied to the human body for cleansing, beautifying, promoting attractiveness, or altering appearance. These compositions provide non-therapeutic benefits and are not regulated as pharmaceuticals. However, in some situations, cosmetic compositions are incorporated into pharmaceutical compositions to provide cosmetic benefits (e.g., products that treat skin or hair diseases, but also contain cosmetic compositions for their coloring or other benefits). Cosmetic compositions include makeup compositions as defined herein. Also, it is intended that the present invention encompass the use of cosmetics on animals other than humans.

As used herein, the terms “pharmaceutical compositions” and “therapeutic compositions” refer to compositions such as drugs that provide medical benefits, rather than solely cosmetic benefits. In the United States, pharmaceutical and therapeutic compositions are approved by the Food and Drug Administration for treatment and/or prevention of particular conditions.

As used herein, the term “drug” is defined as it is in the FD&C Act definition. Thus, drugs are defined as articles intended for use in the diagnosis, cure, mitigation, treatment or prevention of disease, and articles (other than food) intended to affect the structure or any function of the body of man or other animals.

As used herein, “leave-on” refers to a composition that is applied to a subject and not removed (e.g., cleansed by washing, rinsing, etc.) for a period of typically at least several hours (e.g., 4-12 hours) before the area exposed to the composition is cleansed.

As used herein, a “rinse-off” composition is a composition that is applied and cleansed (e.g., by washing, rinsing, etc.) soon after its application (generally within about 30 minutes of application). In some preferred embodiments, rinse-off compositions are formulated so as to deposit an effective amount of active(s) on the area treated.

As used herein, the term “cosmetic benefit” refers to a desired cosmetic change that results from the administration of a personal care composition. Cosmetic benefits include but are not limited to improvements in the condition of skin, hair, nails, and the oral cavity. In preferred embodiments, at least one cosmetic benefit is provided by the skin care, hair care, nail care, and makeup compositions of the present invention.

As used herein, “cosmetically acceptable” refers to materials that are suitable for use in contact with tissues of humans and/or other animals, without undue toxicity, incompatibility, instability, irritation, allergic responses, etc., commensurate with a reasonable benefit/risk ratio.

As used herein, the terms “pigment,” “color pigment,” and “dye” used in reference to the compositions of the present invention encompasses any compound that provides a color to the composition and/or imparts a color to the surface (e.g., skin and/or hair) to which the composition is applied.

The term “radioprotective” refers to a substance capable of blocking or filtering. UV radiation sunscreens and sunblocks.

As used herein, “improving the appearance and/or condition of skin” refers to any benefit achieved through use of the personal care compositions of the present invention. Examples of benefits include but are not limited to reducing the reducing imperfections and/or blemishes in skin color, including lightening hyperpigmented regions of skin and/or evening skin pigmentation, relieving dryness, eliminating rough, dry spots, improving the skin's ability to retain moisture and/or protect itself from environmental stresses, reducing the appearance of fine lines and wrinkles, improving appearance and skin tone, increasing skin firmness and/or suppleness, decreasing sagging of skin, increasing skin glow and clarity, increasing the skin renewal process, and/or removing vellus hair. Improving the visual appearance of skin also encompasses regulating wrinkles, atrophy, skin lightening, skin darkening, skin smoothness, and/or reducing the visual appearance of pores. In some embodiments, improving the appearance and/or condition of the skin results in skin improvements due to the treatment of a skin disorder with the personal care composition of the invention.

The term “angiogenesis” refers to the biological processes which result in the development of blood vessels and/or increase in the vascularization of tissue in an organism.

The terms “angiogenic disease,” “angiogenic disorder,” and “angiogenic skin disorder,” are used in reference to a disorder, generally a skin disorder or related disorder which occurs as a consequence of or which results in increased vascularization in tissue. Oftentimes, the etiology of the angiogenic disease is unknown. However, whether angiogenesis is an actual cause of a disease state or is simply a condition of the disease state is unimportant, but the inhibition of angiogenesis in treating or reversing the disease state or condition is an important aspect of the present invention. Thus, it is not intended that the present invention be limited to any particular mechanisms of action. Examples of angiogenic skin disorders which are suitable for treatment utilizing compounds of the present invention include, but are not limited to psoriasis, acne, rosacea, warts, eczema, hemangiomas and lymphangiogenesis, Sturge-Weber syndrome, neurofibromatosis, tuberous sclerosis, chronic inflammatory disease, and arthritis. Any skin disorder which has as a primary or secondary characterization, increased vascularization, is considered an angiogenic skin disorder herein. Thus, the personal care compositions comprising VEGF-binding BBPIs (VEGF-BBPIs) provided by the present invention find use in treatment of a wide variety of angiogenic skin disorders and/or conditions.

The term “rosacea” is used to describe acne, rosacea, or erythematosa characterized by vascular and follicular dilation typically involving the nose and contiguous portions of the cheeks. Rosacea may vary from very mild but persistent erythema to extensive hyperplasia of the sebaceous glands with deep-seated papules and pustules and be accompanied by telangiectasia at the affected erythematous sites. This condition is also referred to as “hypertrophic rosacea” or “rhinophyma,” depending upon the severity of the condition. It is intended that the term encompass all of the various forms of the condition.

The term “psoriasis” is used to describe a skin condition which is characterized by the eruption of circumscribed, discrete and confluent, reddish, silvery-scaled maculopapules. Although it is not intended that the present invention be limited to any particular body area, psoriatic lesions typically occur on the elbows, knees, scalp and trunk. Microscopically, these lesions demonstrate characteristic parakeratosis and elongation of rete ridges.

The term “acne” is used to describe a condition of the skin characterized by inflammatory follicular, papular and pustular eruptions involving the sebaceous apparatus. Although there are numerous forms of acne, the most common form is known as acne simplex or acne vulgaris which is characterized by eruptions of the face, upper back and chest and is primarily comprised of comedones, cysts, papules and pustules on an inflammatory base. The condition occurs primarily during puberty and adolescence due to an overactive sebaceous apparatus which is believed to be affected by hormonal activity.

The term “eczema” is a generic term used to describe acute or chronic inflammatory conditions of the skin, typically erythematous, edematous, papular, vesicular and/or crusting. These conditions are often followed by lichenification, scaling and occasionally, by duskiness of the erythema and, infrequently, hyperpigmentation. Eczema is often accompanied by the sensation of itching and burning. Eczema vesicles form due to intraepidermal spongiosis. Eczema is sometimes referred to colloquially as “tetter,” “dry tetter,” and “scaly tetter.” There are numerous subcategories of eczema, all of which are treated by one or more of the compounds according to the present invention.

The term “hemangioma” refers to a benign self-involuting tumour of endothelial cells (the cells that line blood vessels). Hemangiomas are connected to the circulatory system and filled with blood. The appearance depends on location. If they are on the surface of the skin they look like a ripe strawberry, if they are just under the skin they present as a bluish swelling. Sometimes they grow in internal organs such as the liver or larynx. Approximately 80% are located on the face and neck, with the next most prevalent location being the liver. The personal care compositions of the invention are intended to treat hemangiomas of the skin i.e. hemangiomas that are on the surface of the skin and hemangiomas that are just under the skin.

The term “scleroderma” herein refers to a chronic disease characterized by excessive deposits of collagen in the skin or other organs. Scleroderma affects the skin, and in more serious cases it can affect the blood vessels and internal organs. The most evident symptom is usually the hardening of the skin and associated scarring. The skin may appear tight, reddish or scaly. Blood vessels may also be more visible. A significant player in the process is transforming growth factor (TGFβ). Topical treatment for the skin changes of scleroderma do not alter the disease course, but may improve pain and ulceration.

As used herein, “hair care composition” refers to compositions that are applied to hair to provide beneficial properties such as thickening, thinning, coloring, decoloring, cleansing, conditioning, softening, shaping, etc. In some embodiments, the hair care composition is in a form selected from the group consisting of shampoos, conditioners, anti-dandruff treatments, styling aids, styling conditioners, hair repair or treatment sera, lotions, creams, pomades, and chemical treatments. In other embodiments, the styling aids are selected from the group consisting of sprays, mousses, rinses, gels, foams, and combinations thereof. In further embodiments, the chemical treatments are selected from the group consisting of permanent waves, relaxers, and permanents, semi-permanents, temporary color treatments and combinations thereof.

As used herein, “inhibiting hair growth” and “inhibition of hair growth” refer to an observed lessening of hair length and/or thickness. Thus, in some preferred embodiments, application of a personal care composition of the present invention provides a benefit in lessening hair length and/or thickness as compared to an area in which a personal care composition of the present invention has not been applied. In some embodiments, the observed reduction of hair growth and/or thickness is a range from less than 1% to more than 99%, as compared to untreated areas, while in other embodiments, the observed reduction is from about 100% to about 90%, from about 90% to about 80%, from about 80% to about 70%, from about 70% to about 60%, from about 60% to about 50%, from about 50% to about 40%, from about 40% to about 30%, from about 30% to about 20%, from about 20% to about 10%, from about 10% to about 1%. Indeed, it is not intended that the term be limited to any particular percentage reduction, as long as the reduction is observable by visual (i.e., by eye) or other means. It is also intended that the term encompass “preventing hair growth” to any degree, as described above. It is not intended that the term be limited to the complete prevention of hair growth (i.e., there is no observed growth of hair).

The terms “dermatological inflammatory disorder” or “inflammatory skin disorder” refer to skin condition associated with inflammation of the skin. In some embodiments, the inflammatory skin disorder is a disorder associated with elevated levels of inflammatory cytokines e.g. TNFα.

As used herein, in some embodiments, the term “compound” refers to the BBPI comprised in the personal care compositions of the invention.

The term “effective amount” is used throughout the specification to describe concentrations or amounts of compounds according to the present invention which may be used to produce a favorable change in the disease or condition treated, e.g. whether that change is hair growth, prevention of hair growth or for ameliorating a condition caused or associated with a disorder. As used herein, “safe and effective amount” refers to a sufficient amount of a material that significantly induces a positive modification to the area upon which the material is applied and also does not result in the production of serious side effects (at a reasonable risk/benefit ratio). The safe and effective amount of the material may vary with the particular skin or other body part being treated, the age of the subject being treated, the severity of the condition being treated, the duration of treatment, the nature of concurrent therapy, the specific material used, the particular carrier utilized, etc. Those of skill in the art are capable of adjusting the concentration of the personal care compositions provided herein for the desired application of the compositions.

As used herein, “active” (and “actives”) refers to a composition that imparts a benefit to a subject being treated. For example, in some embodiments, the present invention provides personal care compositions comprising a modified variant BBPI, e.g. modified variant VEGF-BBPI, a “primary active” which functions to provide benefit to the area to which it is applied. Thus, in some embodiments, the modified variant VEGF-BBPI is present in skin care formulations and serves to treat the skin of subjects suffering from an angiogenic skin disorder. It is not intended that the term be limited to VEGF-BBPI, as there are additional constituents present in the personal care compositions of the present invention which impart benefits. In some embodiments, these additional constituents are encompassed by the designation “secondary actives.” Primary and secondary actives are collectively referred to as “actives” herein. Other “primary actives” provided by the invention include FGF-BBPIs, TGF-BBPIs and TNF-BBPIs.

As used herein, “vitamin B₃ compound” means a compound having the formula:

wherein R is —CONH₂ (i.e., niacinamide), —COOH (i.e., nicotinic acid) or —CH₂OH (i.e., nicotinyl alcohol); derivatives thereof; and salts of any of the foregoing.

As used herein, “non-vasodilating” means that an ester does not commonly yield a visible flushing response after application to the skin in the subject compositions. It is contemplated that the majority of the general population would not experience a visible flushing response, although such compounds may cause vasodilation not visible to the naked eye.

As used herein, “retinoid” includes all natural and/or synthetic analogs of Vitamin A and/or retinol-like compounds which possess the biological activity of Vitamin A in/on the skin, as well as the geometric isomers and stereoisomers of these compounds. However, it is not intended that the term be limited to these compounds, as the term encompasses vitamin A alcohol (retinol) and its derivatives such as vitamin A aldehyde (retinal), vitamin A acid (retinoic acid) and vitamin A esters (e.g., retinyl acetate, retinyl propionate and retinyl palmitate), etc. It is further intended that the term encompass all-trans-retinoic acids and 13-cis-retinoic acids. It is also intended that the term encompass compositions that are encapsulated, as well as provided for use in various forms. The terms “retinol” and “retinal” preferably comprise the all-trans compounds. The retinoid preferably used for the formulation of the present invention is all-trans-retinol, generally referred to as “retinol” herein.

As used herein, “carotenoid” is used in reference to β-carotene, lycopene, lutein, astaxanthin, zeaxanthin, cryptoxanthin, citranaxanthin, canthaxanthin, bixin, β-apo-4-carotenal, β-apo-8-carotenal, β-apo-8-carotenoic esters, alone, as well as in combination. Carotenoids which are preferably used are β-carotene, lycopene, lutein, astaxanthin, zeaxanthin, citranaxanthin and canthaxanthin. In some embodiments, carotenoids are utilized in crystalline form, as well as in formulations, including but not limited to dry powders (See e.g., dry powders, as described in EP 0 065 193; hereby incorporated by reference). In some embodiments, the preferred use in the case of lycopene, astaxanthin and canthaxanthin is of lycopene-, astaxanthin- and canthaxanthin-containing dry powders, for example LYCOVIT®, LUCANTIN® Pink and LUCANTIN® Red (10% dry powders respectively of lycopene, astaxanthin and canthaxanthin, commercially available from BASF AG, Ludwigshafen, Germany. As used herein, the term “dispersed phase” is used as by those of skill in the art of emulsion technology as the phase that exists as small particles or droplets suspended in and surrounded by a continuous phase. The dispersed phase is also known as the “internal” or “discontinuous” phase.

As used herein, “penetration enhancers” refer to compositions that facilitate penetration through the upper stratum corneum barrier to the deeper skin layers. Examples of penetration enhancers include, but are not limited to, propylene glycol, azone, ethoxydiglycol, dimethyl isosorbide, urea, ethanol, dimethyl sulfoxide, microemulsions, liposomes, and nanoemulsions.

As used herein, the terms “emulsifier” and “surfactant” refer to compounds that disperse and suspend the dispersed phase within the continuous phase of a material. Surfactants find particular use in products intended for skin and/or hair cleansing. In particular embodiments, the term “surfactant(s)” is used in reference to surface-active agents, whether used as emulsifiers or for other surfactant purposes such as skin cleansing.

In various embodiments, the present invention also includes “protectants” such as UV absorbers (e.g., octyl methoxycinnamate, benzophenone-3, titanium dioxide, and octyl salicylate); film-forming agents (e.g., VP/Eicosene copolymer); cosmeceutical agents (e.g., peptides and proteins, alpha hydroxy acids, and retinol and retinoic acid derivatives); antioxidants (e.g., tocopherol and derivatives thereof and ascorbic acid and derivatives thereof); vitamins (e.g., B, D, K and their derivatives); antiperspirant actives (e.g., aluminum hydroxide and zirconium hydroxide); depilating agents (e.g., thioglycolate salts); anti-acne agents (e.g., salicylic acid and benzoyl peroxide); abrasives and exfoliants (e.g., silicates, pumice, and polyethylene); and extracts of plant, fruit, vegetable and/or marine sources.

As used herein, the term “bioactivity” refers to a cause and effect relationship between a composition and a biological system. Thus, the term is used as by those skilled in the art of biotechnology and biological sciences as the phrase that describes a cause and effect relationship between a molecular composition and living biological matter (e.g., tissue, cells, etc.).

As used herein as a noun, the term “bioactive” refers a composition that exhibits bioactivity upon administration to living biological matter (e.g., tissue, cells, etc.). The term is used synonymously with “bioactive compound.”

As used herein, “silicone gum” means high molecular weight silicones having an average molecular weight in excess of about 200,000 and preferably from about 200,000 to about 4,000,000. It is intended that the definition encompass non-volatile polyalkyl and polyaryl siloxane gums.

As used herein, a “composition comprising a modified variant BBPI” refers broadly to any composition containing the given modified variant BBPI. The composition may be in any form, particularly a form that is suitable for administration.

As used herein, a compound is said to be “in a form suitable for administration” when the compound may be administered to a human or other animal by any desired route (e.g., topical, oral, etc.).

As used herein, “safe and effective amount” refers to a sufficient amount of a material that significantly induces a positive modification to the area upon which the material is applied and also does not result in the production of serious side effects (at a reasonable risk/benefit ratio). The safe and effective amount of the material may vary with the particular skin or other body part being treated, the age of the subject being treated, the severity of the condition being treated, the duration of treatment, the nature of concurrent therapy, the specific material used, the particular carrier utilized, etc. Those of skill in the art are capable of adjusting the concentration of the personal care compositions provided herein for the desired application of the compositions.

5.2 BOWMAN BIRK PROTEASE INHIBITOR SCAFFOLDS

Bowman Birk Protease Inhibitor proteins (BBPI) are a kinetically and structurally well-characterized family of small proteins (60-90 residues), and have been found only in the seed of monocotyledous and dicotyledonous plants, and have not been identified in any other part of the plant (See e.g., Birk, Int. J. Pept. Protein Res., 25:113-131 [1985]). The sequences of many wild-type BBPI scaffolds have been determined from both monocotyledonous and dicotyledonous seeds, and have been analyzed (Prakash et al., J mol Evol 42:560-569 [1996]). They typically have a symmetrical structure of two tricyclic domains each containing an independent binding loop, although some have one domain and some have more than two domains. The major ˜8 kDa Bowman Birk Inhibitor isolated from soybeans (BBI) has two separate reactive site loops, loop I inhibits proteases having trypsin-like specificity and loop 11 inhibits proteases with chymotrypsin-like specificity (See e.g., Chen et al., J. Biol. Chem., 267:1990-1994 [1992]; Werner and Wemmer, Biochem., 31:999-1010 [1992]; Lin et al., Eur. J. Biochem., 212:549-555; Voss et al., Eur. J. Biochem., 242:122-131 [1996]; and Billings et al., Pro. Natl. Acad. Sci., 89:3120-3124 [1992]). These binding regions each contain a “canonical loop” structure, which is a motif found in a variety of serine proteinase inhibitors (Bode and Huber, Eur. J. Biochem., 204:433-451 [1992]). In some embodiments, wild-type BBPI scaffolds serve as wild-type precursor BBPI scaffolds from which variant and modified variant BBPI scaffolds are derived.

In some embodiments, the present invention provides for variant BBPI scaffolds in which the trypsin (loop I) and/or chymotrypsin loop(s) (loop II) is replaced by a variant peptide. In some embodiments, the trypsin loop is replaced with a variant peptide resulting in a variant BBPI that retains chymotrypsin inhibitory activity (CIA). In other embodiments, the chymotrypsin loop is replaced with a variant peptide to generate a variant BBPI that retains trypsin inhibitory activity (TIA). In yet other embodiments, both the trypsin and the chymotrypsin loops of the BBPI are each replaced with a variant peptide. Non-limiting examples of BBPI scaffolds in which the trypsin and/or chymotrypsin loop is replaced with a variant peptide sequence include wild-type and unmodified variant scaffolds. Examples of wild-type precursor scaffolds include but are not limited to the scaffolds disclosed by Prakash (supra), such as the scaffold of the soybean inhibitor from Glycine max

(BBI; SEQ ID NO:13) or the mature and truncated form thereof (SEQ ID NO:185; DDESSKPCCDQCACTKSNPPQCRCSDMRLNSCHSACKSCICALSYPAQCFCVDITDFCYEPCKPSE), the inhibitor from Dolichos biflorus (BBdb; SEQ ID NO:449; PSESSKPCCDQCACTKSIPPQCRCTDVRLNSCHSACSSCVCTFSIPAQCVCVDMKDFCYEPCK; the soybean inhibitor D-II from Glycine max (BBsb3; SEQ ID NO:450; DDEYSKPCCDLCMCTRSMPPQCSCEDIRLNSCHSDCKSCMCTRSQPGQCRCLDTNDFCYKPCKSRDD) and the inhibitor from Torresea (Amburana) cearensis (BBtc; SEQ ID NO:451; SSKWEACCDRCACTKSIPPQCHCADIRLNSCHSACESCACTHSIPAQCRCFDITDFCYKPCSG). Examples of unmodified variant precursor scaffolds include but are not limited to scaffolds in which the chymotrypsin loop has been replaced with a VEGF binding variant sequence include the

BBI-AV (SEQ ID NO:186; DDESSKPCCDQCACTKSNPPQCRCSDMRLNSCHSACKSCACYNLYGWTCFCVDITDFCYEPCKPSEDDKEN), BBIt-AV (SEQ ID NO:187; DDESSKPCCDQCACTKSNPPQCRCSDMRLNSCHSACKSCACYNLYGWTCFCVDITDFCYEPCKPSE), BBdb-AV (SEQ ID NO:452; DPSESSKPCCDQCACTKSIPPQCRCTDVRLNSCHSACSSCACYNLYGWTCVCVDMKDFCYEPCK), BBsb3-AV (SEQ ID NO:453; DPDDEYSKPCCDLCMCTRSMPPQCSCEDIRLNSCHSDCKSCACYNLYGWTCRCLDTNDFCYKPCKSRDD), and BBtc-AV (SEQ ID NO:454; DPSSKWEACCDRCACTKSIPPQCHCADIRLNSCHSACESCACYNLYGWTCRCFDITDFCYKPCSG), BBIt-VEGK (SEQ ID NO:640; DPDDESSKPCCDQCICTKSNPPQCRCRDARPNACHSACKSCACKYYLYWWCKCTDITDFCYEPCKPSE), BBIt-VEGT (SEQ ID NO:641; DPDDESSKPCCDQCICTKSNPPQCRCRDARPNACHSACKSCACTLWKSYWCKCTDITDFCYEPCKPSE) and

BBIt-VEGKD (SE ID NO:642; DPDDESSKPCCDQCICTKSNPPQCRCRDARPNACHSACKSCACKYDLYWWCKCTDITDFCYEPCKPSE). In some embodiments, unmodified variant precursor scaffolds are variant scaffolds in which a variant peptide replaces the chymotrypsin loop of the wild-type BBPI and which also introduces a substitution of the amino acid at the position equivalent to position 40 of the BBPI of SEQ ID NO:187. For example, the unmodified variant BBPI scaffold of SEQ ID NO:187 (BBIt-AV) was derived from the wild-type precursor scaffold of SEQ ID NO:185 by replacing the chymotrypsin loop of SEQ ID NO:185 with the VEGF variant peptide of SEQ ID NO:9, which introduces an amino acid substitution I40A in addition to replacing the chymotrypsin loop. The unmodified variant BBPI of SEQ ID NO:187 is modified to generate a modified variant BBPI, which in addition to the replaced chymotrypsin loop, comprises at least one amino acid substitution as described below.

Although numerous isoforms of BBI have been characterized, SEQ ID NO:13 is an example of the amino acid sequence of the wild-type BBI scaffold used in some embodiments comprising approximately 71 amino acid residues (See Example 1). In some embodiments, the invention provides for BBPI scaffolds e.g. SEQ ID NO:11, that include the pro region, while in other embodiments, the invention provides for BBI scaffolds from which the pro peptide has been removed e.g. SEQ ID NO:185. In yet other embodiments, the invention provides for BBI scaffolds from which up to 10 amino acids have been removed from the N- or C-terminus. In some embodiments, the invention provides for BBI scaffolds from which up to 5 e.g. SEQ ID NO:187. It will be appreciated that truncations of the BBI scaffold will not destroy the ability of the BBI to bind the target protein.

In soybeans, BBPIs e.g. BBI is produced as a pro-protein with an N-terminal pro-peptide that is 19 amino acids in length. Thus, in some embodiments, BBI is produced with all or at least a portion of the propeptide. In some embodiments, BBI is truncated, with as many as 10 amino acid residues being removed from either the N- or C-terminal. For example, upon seed desiccation, some BBPI molecules have the C-terminal 9 or 10 amino acid residues removed. Thus, proteolysis is generally highly tolerated prior to the initial disulfide and just after the terminal disulfide bond, the consequences of which are usually not detrimental to the binding to target protein. However, it will be appreciated that any one of the isoforms or truncated forms a BBPI find use in various embodiments of the present invention. In some embodiments, the truncated form of BBPI that finds use in the present invention is the variant BBIt in which the chymotrypsin loop is replaced with a variant sequence as described below.

5.3 VARIANT BBPIS

As indicated above, BBPIs have binding loops (i.e. trypsin and chymotrypsin loops) that inhibit proteases. The present invention provides variant BBPIs, which are derived from wild-type or from unmodified variant BBPI precursor scaffolds in which one or more reactive sites (e.g., Loop I (trypsin) and/or Loop II (chymotrypsin) of BBPIs have been replaced with variant peptides that bind a target protein. Non-limiting examples of target proteins that are bound by variant peptides comprised in the BBPIs of the invention include various cytokines, including cytokines of the tumor necrosis factor (TNF) family, particularly TNF-α; cytokines of the transforming growth factor family, particularly TGFβ; cytokines of the fibroblast growth factor family (FGF), particularly FGF-5, and cytokines of the vascular endothelial growth factor (VEGF) family, particularly VEGF-A. In addition, variant peptides that replace one or both loops of the BBPIs of the invention include peptides that interact with inhibitors of the complement pathway such as C2, C3, C4 or C5 inhibitors, Compstatin, and other proteins of interest. Indeed, it is not intended that the present invention be limited to any particular sequence substituted into either of these loops, as any suitable sequence finds use in the present invention.

In some embodiments, the trypsin and/or chymotrypsin loop(s) of the BBPI precursor scaffold is replaced with variant sequence that binds VEGF to generate variant VEGF-BBPI proteins. In some embodiments, the variant BBPI is derived from a wild-type or an unmodified variant BBPI precursor scaffold chosen from the scaffolds of the soybean inhibitor from Glycine max (BBI; SEQ ID NO:13) or the mature and truncated form thereof (SEQ ID NO:185), the inhibitor from Dolichos biflorus (BBdb; SEQ ID NO:449), the soybean inhibitor D-II from Glycine max (BBsb3; SEQ ID NO:450), the inhibitor from Torresea (Amburana) cearensis (BBtc; SEQ ID NO:451), the BBI-AV scaffold of (SEQ ID NO:186), the BBIt-AV scaffold of (SEQ ID NO:187), the BBdb-AV scaffold of (SEQ ID NO:452), the BBsb3-AV scaffold of (SEQ ID NO:453), the BBtc-AV scaffold of (SEQ ID NO:454), the BBIt-VEGK scaffold of (SEQ ID NO:640), the BBIt-VEGT scaffold of (SEQ ID NO:641) and the BBIt-VEGKD scaffold of (SE ID NO:642). In addition, any wild-type BBPI precursor scaffolds, such as those disclosed by Prakash et al. (supra), may be used to generate variant BBPI scaffolds.

In some embodiments, the VEGF variant sequences include, but are not limited to VEGF-binding peptides disclosed in U.S. application Ser. Nos. 09/832,723 and 10/984,270, including peptides ACYNLYGWTC (SEQ ID NO:9), KYYLYWW (SEQ ID NO:458), TLWKSYW (SEQ ID NO:459), DLYWW (SEQ ID NO:460), SKHSQIT (SEQ ID NO:468) KTNPSGS (SEQ ID NO:469) RPTGHSL (SEQ ID NO:470), KHSAKAE (SEQ ID NO:471) KPSSASS (SEQ ID NO:472), PVTKRVH (SEQ ID NO:473), TLHWWVT (SEQ ID NO:492), PYKASFY (SEQ ID NO:493), PLRTSHT (SEQ ID NO:494), EATPROT (SEQ ID NO:495), NPLHTLS (SEQ ID NO:496), KHERIWS (SEQ ID NO:497), ATNPPPM (SEQ ID NO:498), STTSPNM (SEQ ID NO:499), ADRSFRY (SEQ ID NO:500), PKADSKQ (SEQ ID NO:501), PNQSHLH (SEQ ID NO:502), SGSETWM (SEQ ID NO:503), ALSAPYS (SEQ ID NO:504), KMPTSKV (SEQ ID NO:505), ITPKRPY (SEQ ID NO:506), KWIVSET (SEQ ID NO:507), PNANAPS (SEQ ID NO:508), NVQSLPL (SEQ ID NO:509), TLWPTFW (SEQ ID NO:510), NLWPHFW (SEQ ID NO:511), SLWPAFW (SEQ ID NO:512), SLWPHFW (SEQ ID NO:513), APWNSHI (SEQ ID NO:514), APWNLHI (SEQ ID NO:515), LPSWHLR (SEQ ID NO:516), PTILEWY (SEQ ID NO:517), TLYPQFW (SEQ ID NO:518), and HLAPSAV (SEQ ID NO:519). In some other embodiments, the VEGF variant sequences include, but are not limited to VEGF-binding peptides disclosed in U.S. application Ser. No. 11/919,717, including peptides KYYLSWW (SEQ ID NO:520), WYTLYKW (SEQ ID NO:521), TYRLYWW (SEQ ID NO:522), RYSLYYW (SEQ ID NO:523), YYLYYWK (SEQ ID NO:524), NYQLYGW (SEQ ID NO:525), TKWPSYW (SEQ ID NO:226), TLWKSYW (SEQ ID NO:527), PLWPSYW (SEQ ID NO:528), RLWPSYW (SEQ ID NO:529), TLWPKYW (SEQ ID NO:530), KYDLYWW (SEQ ID NO;531), RYDLYWW (SEQ ID NO:532), DYRLYWW (SEQ ID NO:533), DYKLYWW (SEQ ID NO:534), EYKLYWW (SEQ ID NO:535), and RYPLYWW (SEQ ID NO:536).

In some embodiments, the trypsin and/or chymotrypsin loop(s) of the BBPI precursor scaffold is replaced with variant sequences that interact with FGF5 to generate variant FGF-BBPI proteins. In some embodiments, the variant BBPI is derived from a wild-type or an unmodified variant BBPI precursor scaffold chosen from the scaffolds of the soybean inhibitor from Glycine max (BBI; SEQ ID NO:13) or the mature and truncated form thereof (SEQ ID NO:185), the inhibitor from Dolichos biflorus (BBdb; SEQ ID NO:449), the soybean inhibitor D-II from Glycine max (BBsb3; SEQ ID NO:450), the inhibitor from Torresea (Amburana) cearensis (BBtc; SEQ ID NO:451), the BBI-AV scaffold of (SEQ ID NO:186), the BBIt-AV scaffold of (SEQ ID NO:187), the BBdb-AV scaffold of (SEQ ID NO:452), the BBsb3-AV scaffold of (SEQ ID NO:453), the BBtc-AV scaffold of (SEQ ID NO:454), the BBIt-VEGK scaffold of (SEQ ID NO:640), the BBIt-VEGT scaffold of (SEQ ID NO:641) and the BBIt-VEGKD scaffold of (SE ID NO:642). In addition, any wild-type BBPI precursor scaffolds, such as those disclosed by Prakash et al. (supra), may be used to generate variant BBPI scaffolds.

In some embodiments, the trypsin and/or chymotrypsin loop(s) of the BBPI precursor scaffold is replaced with variant sequences that interact with FGF5. In some embodiments, the FGF5 variant sequences include, but are not limited to FGF5-binding peptides disclosed in U.S. application Ser. Nos. 10/984,410 and 12/033,848, including peptides CACRTQPYPLCF (MM007; SEQ ID NO:430), CICTWIDSTPC(PS2; SEQ ID NO:431), CYGLPFTRC (SEQ ID NO:537), CEEIWTMLC (SEQ ID NO:538), CWALTVKTC (SEQ ID NO:539), CLTVLWTTC (SEQ ID NO:540), CTLWNRSPC (SEQ ID NO:541), CHYLLTNYC (SEQ ID NO:542), CRIHLAHKC (SEQ ID NO:543), TNIDSTP(SEQ ID NO:544), HLQTTET (SEQ ID NO:545), SLNNLTV (SEQ ID NO:546), TNIDSTP (SEQ ID NO:547), TNIDSTP (SEQ ID NO:548), LRILANK (SEQ ID NO:549), LLTPTLN (SEQ ID NO:550), ALPTHSN (SEQ ID NO:551), TNIDSTP (SEQ ID NO:552), LCRRFEN (SEQ ID NO:553), TNIDSTP (SEQ ID NO:554), TNIDSTP (SEQ ID NO:555), HLQTTET (SEQ ID NO:556), PLGLCPP (SEQ ID NO:557), GYFIPSI (SEQ ID NO:558), TKIDSTP (SEQ ID NO:559), HLQTTET (SEQ ID NO:560), WNIDSTP (SEQ ID NO:561), TWIDWTP (SEQ ID NO:562), RTQPYPL (SEQ ID NO:670) and TWIDSTP (SEQ ID NO:671).

In some embodiments, the trypsin and/or chymotrypsin loop(s) of the BBPI precursor scaffold is replaced with variant sequences that interact with TGFβ to generate variant TGF-BBPIs. In some embodiments, the variant BBPI is derived from a wild-type or an unmodified variant BBPI precursor scaffold chosen from the scaffolds of the soybean inhibitor from Glycine max (BBI; SEQ ID NO:13) or the mature and truncated form thereof (SEQ ID NO:185), the inhibitor from Dolichos biflorus (BBdb; SEQ ID NO:449), the soybean inhibitor D-II from Glycine max (BBsb3; SEQ ID NO:450), the inhibitor from Torresea (Amburana) cearensis (BBtc; SEQ ID NO:451), the BBI-AV scaffold of (SEQ ID NO:186), the BBIt-AV scaffold of (SEQ ID NO:187), the BBdb-AV scaffold of (SEQ ID NO:452), the BBsb3-AV scaffold of (SEQ ID NO:453), the BBtc-AV scaffold of (SEQ ID NO:454), the BBIt-VEGK scaffold of (SEQ ID NO:640), the BBIt-VEGT scaffold of (SEQ ID NO:641) and the BBIt-VEGKD scaffold of (SE ID NO:642). In addition, any wild-type BBPI precursor scaffolds, such as those disclosed by Prakash et al. (supra), may be used to generate variant BBPI scaffolds.

In some embodiments, the trypsin and/or chymotrypsin loop(s) of the BBPI precursor scaffold is replaced with variant sequences that interact with TGFβ. In some embodiments, the TGFβ variant sequences include, but are not limited to TGFβ-binding peptides disclosed in U.S. application Ser. No. 10/581,142, including peptides CLCPENINVLPCN (PEN3; SEQ ID NO:436), CICKHNVDWLCF (MMO21W; SEQ ID NO:437), CICWTQHIHNCF (WTQ; SEQ ID NO:438), CVTTDWIEC (SEQ ID NO:563), CYYSQFHQC (SEQ ID NO:564), CPTLWTHMC (SEQ ID NO:565), QSACIVYYVGRKPKVECASSD (SEQ ID NO:566), QSACILYYIGKTPKIECASSD (SEQ ID NO:567), QSACILYYVGRTPKVECASSD (SEQ ID NO:568), acetyl-LCPENDNVSPCY-cohn2 (SEQ ID NO:569), KHNVRLL (SEQ ID NO:570), NDTPSYF (SEQ ID NO:571), AKLYAGS (SEQ ID NO:572), RGPAHSL (SEQ ID NO:573), NSLAERR (SEQ ID NO:574), HPLASPH (SEQ ID NO:575), QPWNKLK (SEQ ID NO:576), AWLr/Mipy (SEQ ID NO:577), PTKPAQQ (SEQ ID NO:578), PSLNRPQ (SEQ ID NO:579), HHARQEW (SEQ ID NO:580), RHHTPGP (SEQ ID NO:581), ASAINPH (SEQ ID NO:582), CHGYDRAPC (SEQ ID NO:644), CFAPADQAC (SEQ ID NO:645), CIPSRFITC (SEQ ID NO:646), CHGHTKLAC (SEQ ID NO:647), CNGKSKLAC (SEQ ID NO:648), PENINVLP (SEQ ID NO;672), KHNVDWL (SEQ ID NO:673) and WTQHIHNC (SEQ ID NO:674).

In some embodiments, the trypsin and/or chymotrypsin loop(s) of the BBPI precursor scaffold is replaced with variant sequences that interact with TNFα to generate variant TNF-BBPIs. In some embodiments, the variant BBPI is derived from a wild-type or an unmodified variant BBPI precursor scaffold chosen from the scaffolds of the soybean inhibitor from Glycine max (BBI; SEQ ID NO:13) or the mature and truncated form thereof (SEQ ID NO:185), the inhibitor from Dolichos biflorus (BBdb; SEQ ID NO:449), the soybean inhibitor D-II from Glycine max (BBsb3; SEQ ID NO:450), the inhibitor from Torresea (Amburana) cearensis (BBtc; SEQ ID NO:451), the BBI-AV scaffold of (SEQ ID NO:186), the BBIt-AV scaffold of (SEQ ID NO:187), the BBdb-AV scaffold of (SEQ ID NO:452), the BBsb3-AV scaffold of (SEQ ID NO:453), the BBtc-AV scaffold of (SEQ ID NO:454), the BBIt-VEGK scaffold of (SEQ ID NO:640), the BBIt-VEGT scaffold of (SEQ ID NO:641) and the BBIt-VEGKD scaffold of (SE ID NO:642). In addition, any wild-type BBPI precursor scaffolds, such as those disclosed by Prakash et al. (supra), may be used to generate variant BBPI scaffolds.

In some embodiments, the trypsin and/or chymotrypsin loop(s) of the BBPI precursor scaffold is replaced with variant sequences that bind TNFα. In some embodiments, the TNFα binding sequences include, but are not limited to TNF-binding peptides disclosed in U.S. application Ser. No. 10/968,732, including peptides RYWQDIP (T1; SEQ ID NO:474), APEPILA (T2; SEQ ID NO:475), DMIMVSI (T3; SEQ ID NO:476), WTPKPTQ (SEQ ID NO:583), ATFPNQS (SEQ ID NO:584), ASTVGGL (SEQ ID NO:585), TMLPYRP (SEQ ID NO:586), AWHSPSV (SEQ ID NO:587), TQSFSS (SEQ ID NO:588), THKNTLR (SEQ ID NO:589), GQTHFHV (SEQ ID NO:590), LPILTQT (SEQ ID NO:591), SILPVSH (SEQ ID NO:592), SQPIPI (SEQ ID NO:593), and QPLRKLP (SEQ ID NO:594).

In some embodiments, the variant BBPIs further comprises a peptide insert that is positioned at the N-terminus of the modified variant BBPI. In some embodiments, the peptide insert comprises a sequence of between 1 and 15 amino acids. In other embodiments, the peptide insert comprises a sequence between 5 and 10 amino acids. In some embodiments, the peptide insert comprises the peptide of SEQ ID NO:389 (DDEPSKPCCDPDP; SEQ ID NO:389). Examples of modified variant BBPIs that comprise the peptide insert of SEQ ID NO:389 are the modified variant 4D13BBIt-AV of (DDEPSKPCCDPDPDDESSKPCCDQCACTKSNPPQCRCSDMRLNSCHSACKSCACY NLYGWTCFCVDITDFCYEPCKPSE; SEQ ID NO:390), and the modified variant BBIt-AV-4D13-13I-29P-40K-50T-52A of SEQ ID NO: 413 (DPDDEPSKPCCDPDPDDESSKPCCDQCICTKSNPPQCRCSDMRPNSCHSACKSCKC YNLYGWTCTCADITDFCYEPCKPSE; SEQ ID NO:413).

In some embodiments, variant sequences are selected by various methods known in the art, including but not limited to phage display and other suitable screening methods. For example, a random peptide gene library is fused with phage PIII gene so the peptide library will be displayed on the surface of the phage. Subsequently, the phage display library is exposed to the target protein and washed with buffer to remove non-specific binding (this process is sometimes referred to as panning). Finally, the binding phage and PCR the DNA sequence for the peptide encoded are isolated.

In most embodiments, one of the loops is replaced with a variant sequence i.e., peptides often 3 to 14 amino acids in length, with 5 to 10 amino acids being preferred, to generate the variant BBPI. Longer sequences find use in the present invention, as long as they provide the binding and/or inhibition desired. In addition, peptides suitable for use as replacements of the binding loop(s) preferably adopt a functional conformation when contained within a constrained loop (i.e., a loop formed by the presence of a disulfide bond between two cysteine residues). In some specific embodiments, the peptides are between 7 and 9 amino acids in length. In other embodiments, the variant sequences are peptides of 10 amino acids in length.

5.4 MODIFIED VARIANT BBPI PROTEINS 5.4.1 Modified Variant BBPIs: Single Amino Acid Substitutions

In some embodiments, the invention provides for modified variant BBPIs, which are variant BBPIs that further comprise at least one amino acid substitution in the backbone of the BBPI. Thus, in some embodiments, modified variant BBPIs are variant BBPIs that contain trypsin and/or chymotrypsin loop(s) that have been replaced by a variant sequence that binds to a target protein, and that are further altered by comprising at least one amino acid substitution C-terminal and/or N-terminal to the replaced loop. Thus, in some embodiments, modified variant BBPIs are the variant BBPIs described in section 5.4 in which the trypsin and/or chymotrypsin loop(s) has been replaced by a variant peptide, but that further comprise a substituted amino acid at least at one position equivalent to a position chosen from positions equivalent to positions 1, 4, 5, 11, 13, 18, 25, 27, 29, 31, 38, 40, 50, 52, 55, and 65 of SEQ ID NO:187, as described in section 5.4.

An amino acid residue of a modified variant BBPI is at an equivalent to the position of a residue of a precursor BBPI if it is homologous (i.e. corresponding in position in primary structure) to a specific residue. In order to establish homology to primary structure, the amino acid sequence of a precursor BBPI is directly compared to the primary amino acid sequence, and particularly to the set of cysteine residues known to be conserved in BBPIs for which the sequence is known. FIG. 17 shows the conserved cysteine residues among exemplary wild type and variant precursor BBPIs described herein. Equivalent residues that are substituted in the modified variant BBPIs of the invention are numbered.

The precursor BBPI may be a naturally-occurring BBPI or a variant BBPI. Specifically, such modified variant BBPIs have an amino acid sequence not found in nature, which is derived by replacement of the trypsin and/or chymotrypsin loop of a precursor BBPI and by replacement of at least one amino acid residue of a precursor BBPI with a different amino acid. In some embodiments, the substitution of the at least one amino acid generates a modified variant BBPI that has a greater protease inhibitory activity than that of the unmodified variant precursor BBPI. In other embodiments, substitution of the at least one amino acid generates a modified variant BBPI that has a greater protease inhibitory activity and production yield than that of the unmodified variant precursor BBPI.

Thus, a modified variant BBPI is derived by substituting at least one amino acid in the backbone of any one variant BBPI scaffold as recited herein. In some embodiments, the isolated modified variant Bowman Birk Protease Inhibitor (BBPI) contains a variant peptide that replaces the chymotrypsin loop of the BBPI scaffold and further comprises a substituted amino acid at least at one amino acid position chosen from positions equivalent to 1, 4, 5, 11, 13, 18, 25, 27, 29, 31, 38, 40, 50, 52, 55, and 65 of the variant BBI of SEQ ID NO:187. In other embodiments, the isolated modified variant Bowman Birk Protease Inhibitor (BBPI) contains a variant peptide that replaces the trypsin loop of the BBPI scaffold and further comprises a substituted amino acid at least at one amino acid position chosen from positions equivalent to 1, 4, 5, 11, 13, 18, 25, 27, 29, 31, 38, 40, 50, 52, 55, and 65 of the variant BBI of SEQ ID NO:187. In yet other embodiments, the isolated modified variant Bowman Birk Protease Inhibitor (BBPI) contains a variant peptide that replaces the trypsin and the chymotrypsin loop of the BBPI scaffold and further comprises a substituted amino acid at least at one amino acid position chosen from positions equivalent to 1, 4, 5, 11, 13, 18, 25, 27, 29, 31, 38, 40, 50, 52, 55, and 65 of the variant BBI of SEQ ID NO:187. In some embodiments, the BBPI scaffold chosen from the scaffolds of the soybean inhibitor from Glycine max (BBI; SEQ ID NO:13) or the mature and truncated form thereof (SEQ ID NO:185), the inhibitor from Dolichos biflorus (BBdb; SEQ ID NO:449), the soybean inhibitor D-II from Glycine max (BBsb3; SEQ ID NO:450), the inhibitor from Torresea (Amburana) cearensis (BBtc; SEQ ID NO:451), the BBI-AV scaffold of (SEQ ID NO:186), the BBIt-AV scaffold of (SEQ ID NO:187), the BBdb-AV scaffold of (SEQ ID NO:452), the BBsb3-AV scaffold of (SEQ ID NO:453), the BBtc-AV scaffold of (SEQ ID NO:454), the BBIt-VEGK scaffold of (SEQ ID NO:640), the BBIt-VEGT scaffold of (SEQ ID NO:641) and the BBIt-VEGKD scaffold of (SEQ ID NO:642). In some embodiments, the variant peptide comprised in the modified variant BBPI is chosen from a VEGF-binding peptide, an FGF-5-binding peptide, a TGFβ-binding peptide and a TNFα-binding peptide. In some embodiments, the VEGF-binding sequences include, but are not limited to VEGF-binding peptides disclosed in U.S. application Ser. Nos. 09/832,723 and 10/984,270, including peptides ACYNLYGWTC (SEQ ID NO:9), KYYLYWW (SEQ ID NO:458), TLWKSYW (SEQ ID NO:459), DLYWW (SEQ ID NO:460), SKHSQIT (SEQ ID NO:468) KTNPSGS (SEQ ID NO:469) RPTGHSL (SEQ ID NO:470), KHSAKAE (SEQ ID NO:471) KPSSASS (SEQ ID NO:472), PVTKRVH (SEQ ID NO:473), TLHWWVT (SEQ ID NO:492), PYKASFY (SEQ ID NO:493), PLRTSHT (SEQ ID NO:494), EATPROT (SEQ ID NO:495), NPLHTLS (SEQ ID NO:496), KHERIWS (SEQ ID NO:497), ATNPPPM (SEQ ID NO:498), STTSPNM (SEQ ID NO:499), ADRSFRY (SEQ ID NO:500), PKADSKQ (SEQ ID NO:501), PNQSHLH (SEQ ID NO:502), SGSETWM (SEQ ID NO:503), ALSAPYS (SEQ ID NO:504), KMPTSKV (SEQ ID NO:505), ITPKRPY (SEQ ID NO:506), KWIVSET (SEQ ID NO:507), PNANAPS (SEQ ID NO:508), NVQSLPL (SEQ ID NO:509), TLWPTFW (SEQ ID NO:510), NLWPHFW (SEQ ID NO:511), SLWPAFW (SEQ ID NO:512), SLWPHFW (SEQ ID NO:513), APWNSHI (SEQ ID NO:514), APWNLHI (SEQ ID NO:515), LPSWHLR (SEQ ID NO:516), PTILEWY (SEQ ID NO:517), TLYPQFW (SEQ ID NO:518), and HLAPSAV (SEQ ID NO:519). In some other embodiments, the VEGF variant sequences include, but are not limited to VEGF-binding peptides disclosed in U.S. application Ser. No. 11/919,717, including peptides KYYLSWW (SEQ ID NO:520), WYTLYKW (SEQ ID NO:521), TYRLYWW (SEQ ID NO:522), RYSLYYW (SEQ ID NO:523), YYLYYWK (SEQ ID NO:524), NYQLYGW (SEQ ID NO:525), TKWPSYW (SEQ ID NO:226), TLWKSYW (SEQ ID NO:527), PLWPSYW (SEQ ID NO:528), RLWPSYW (SEQ ID NO:529), TLWPKYW (SEQ ID NO:530), KYDLYWW (SEQ ID NO;531), RYDLYWW (SEQ ID NO:532), DYRLYWW (SEQ ID NO:533), DYKLYWW (SEQ ID NO:534), EYKLYWW (SEQ ID NO:535), and RYPLYWW (SEQ ID NO:536).

In other embodiments, the FGF5-binding sequences include, but are not limited to FGF5-binding peptides disclosed in U.S. application Ser. Nos. 10/984,410 and 12/033,848, including peptides CACRTQPYPLCF (MM007; SEQ ID NO:430), CICTWIDSTPC(PS2; SEQ ID NO:431), CYGLPFTRC (SEQ ID NO:537), CEEIWTMLC (SEQ ID NO:538), CWALTVKTC (SEQ ID NO:539), CLTVLWTTC (SEQ ID NO:540), CTLWNRSPC (SEQ ID NO:541), CHYLLTNYC (SEQ ID NO:542), CRIHLAHKC (SEQ ID NO:543), TNIDSTP (SEQ ID NO:544), HLQTTET (SEQ ID NO:545), SLNNLTV (SEQ ID NO:546), TNIDSTP (SEQ ID NO:547), TNIDSTP (SEQ ID NO:548), LRILANK (SEQ ID NO:549), LLTPTLN (SEQ ID NO:550), ALPTHSN (SEQ ID NO:551), TNIDSTP (SEQ ID NO:552), LCRRFEN (SEQ ID NO:553), TNIDSTP (SEQ ID NO:554), TNIDSTP (SEQ ID NO:555), HLQTTET (SEQ ID NO:556), PLGLCPP (SEQ ID NO:557), GYFIPSI (SEQ ID NO:558), TKIDSTP (SEQ ID NO:559), HLQTTET (SEQ ID NO:560), WNIDSTP (SEQ ID NO:561), TWIDWTP (SEQ ID NO:562), RTQPYPL (SEQ ID NO:670) and TWIDSTP (SEQ ID NO:671).

In other embodiments, the variant peptide is a TGF-β-binding peptide is chosen from TGFβ-binding sequences that include, but are not limited to TGFβ-binding peptides disclosed in U.S. application Ser. No. 10/581,142, including peptides CLCPENINVLPCN (PEN3; SEQ ID NO:436), CICKHNVDWLCF (MMO21W; SEQ ID NO:437), CICWTQHIHNCF (WTQ; SEQ ID NO:438), CVTTDWIEC (SEQ ID NO:563), CYYSQFHQC (SEQ ID NO:564), CPTLWTHMC (SEQ ID NO:565), QSACIVYYVGRKPKVECASSD (SEQ ID NO:566), QSACILYYIGKTPKIECASSD (SEQ ID NO:567), QSACILYYVGRTPKVECASSD (SEQ ID NO:568), acetyl-LCPENDNVSPCY-cohn2 (SEQ ID NO:569), KHNVRLL (SEQ ID NO:570), NDTPSYF (SEQ ID NO:571), AKLYAGS (SEQ ID NO:572), RGPAHSL (SEQ ID NO:573), NSLAERR (SEQ ID NO:574), HPLASPH (SEQ ID NO:575), QPWNKLK (SEQ ID NO:576), AWLr/Mipy (SEQ ID NO:577), PTKPAQQ (SEQ ID NO:578), PSLNRPQ (SEQ ID NO:579), HHARQEW (SEQ ID NO:580), RHHTPGP (SEQ ID NO:581), ASAINPH (SEQ ID NO:582), CHGYDRAPC (SEQ ID NO:644), CFAPADQAC (SEQ ID NO:645), CIPSRFITC (SEQ ID NO:646), CHGHTKLAC (SEQ ID NO:647), CNGKSKLAC (SEQ ID NO:648), PENINVLP (SEQ ID NO;672), KHNVDWL (SEQ ID NO:673) and WTQHIHNC (SEQ ID NO:674).

In yet other embodiments, the variant peptide is a TNFα-binding peptide is chosen from TNFα binding sequences that include, but are not limited to TNF-binding peptides disclosed in U.S. application Ser. No. 10/968,732, including peptides RYWQDIP (T1; SEQ ID NO:474), APEPILA (T2; SEQ ID NO:475), DMIMVSI (T3; SEQ ID NO:476), WTPKPTQ (SEQ ID NO:583), ATFPNQS (SEQ ID NO:584), ASTVGGL (SEQ ID NO:585), TMLPYRP (SEQ ID NO:586), AWHSPSV (SEQ ID NO:587), TQSFSS (SEQ ID NO:588), THKNTLR (SEQ ID NO:589), GQTHFHV (SEQ ID NO:590), LPILTQT (SEQ ID NO:591), SILPVSH (SEQ ID NO:592), SQPIPI (SEQ ID NO:593), and QPLRKLP (SEQ ID NO:594).

In some embodiments, the at least one amino acid substitution contained at least at one equivalent to a position chosen from positions equivalent to positions 1, 4, 5, 11, 13, 18, 25, 27, 29, 31, 38, 40, 50, 52, 55, and 65 of SEQ ID NO:187 result in the following substituted amino acids. In one embodiment, the substituted amino acid at the amino acid position equivalent to position 1 of SEQ ID NO:187 is chosen from A and C. In another embodiment, the substituted amino acid at the amino acid position equivalent to position 4 of SEQ ID NO:187 is V. In another embodiment, the substituted amino acid at the amino acid position equivalent to position 5 of SEQ ID NO:187 is chosen from P, and A. In another embodiment, the substituted amino acid at the amino acid position equivalent to position 11 of SEQ ID NO:187 is G. In another embodiment, the substituted amino acid at the amino acid position equivalent to position 13 of SEQ ID NO:187 is chosen from Y, I, F, M, L, V, K, and R. In another embodiment, the substituted amino acid at the amino acid position equivalent to position 18 of SEQ ID NO:187 include 1, V and L. In another embodiment, the substituted amino acid at the amino acid position equivalent to position 25 of SEQ ID NO:187 is chosen from K, N, W, I, A and R. In another embodiment, the substituted amino acid at the amino acid position equivalent to position 27 of SEQ ID NO:187 include R, K, V, A, and Q. In another embodiment, the substituted amino acid at the amino acid position equivalent to position 29 of SEQ ID NO:187 is chosen from R, K, and P. In another embodiment, the substituted amino acid at the amino acid position equivalent to position 31 of SEQ ID NO:187 is chosen from Q, H, E, A, R, W, K and T. In another embodiment, the substituted amino acid at the amino acid position equivalent to position 38 of SEQ ID NO:187 is chosen from N, K and R. In another embodiment, the substituted amino acid at the amino acid position equivalent to position 40 of SEQ ID NO:187 is chosen from H, K, Q, R, and Y. In another embodiment, the substituted amino acid at the amino acid position equivalent to position 50 of SEQ ID NO:187 is chosen from R, Q, K, T, V, M, and S. In another embodiment, the substituted amino acid at the amino acid position equivalent to position 52 of SEQ ID NO:187 is chosen from K, T, R, Q, L, H, A, M, S and E. In another embodiment, the substituted amino acid at the amino acid position equivalent to position 55 of SEQ ID NO:187 is M. In another embodiment, the substituted amino acid at the amino acid position equivalent to position 65 of SEQ ID NO:187 is chosen from E, Q, and D. In some embodiments, a single amino acid substitution made in a variant BBPI results in a modified variant BBPI that has a greater protease inhibitory activity than that of the precursor unmodified variant BBPI. In some embodiments, a single amino acid substitution generates a modified variant BBPI that has greater trypsin inhibitory activity (TIA) than the unmodified precursor variant BBPI; while in other embodiments, a single amino acid substitution generates a modified variant BBPI that has greater chymotrypsin inhibitory activity (CIA) than the unmodified precursor variant BBPI.

In one embodiment, the modified variant BBPI is the variant BBI of SEQ ID NO:187 (BBIt-AV; FIG. 9), which contains a VEGF variant peptide in place of the chymotrypsin loop, and which is further modified to contain an amino acid substitution at least at one position chosen from positions 1, 4, 5, 11, 13, 18, 25, 27, 29, 31, 38, 40, 50, 52, 55, and 65 of SEQ ID NO:187 to generate a modified variant BBI. Any one of the single amino acid substitutions described above and made in the variant BBIt-AV BBPI of SEQ ID NO:187 generated modified variant BBIt-AV BBPIs that have greater trypsin inhibitory activity than the unmodified precursor variant BBIt-AV (SEQ ID NO:187; see Example 10).

5.4.2 Modified Variant BBPIs: Combinations of Amino Acid Substitutions

The invention encompasses modified variant BBPIs comprising at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen and at least sixteen amino acid substitutions. In some embodiments, the at least two, at least three, at least four, at least five, at least six, at least seven, at least eight amino, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen and at least sixteen acid substitutions generate modified variant BBPIs that have greater TIA than the unmodified precursor variant BBPI. In other embodiments, the at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, amino acid substitutions generate modified variant BBPIs that have greater TIA and production yield than the unmodified precursor variant BBPI.

In some embodiments, the modified variant BBPI comprises a combination of two amino acid substitutions at amino acids at positions equivalent to positions 50 and 52 of SEQ ID NO:187. In some embodiments, the combination of two amino acid substitutions is 50T-52A. The invention provides for any one of the variant BBPI scaffolds described in Section 5.3 and further comprising the combination of the two amino acid substitutions 50T-52A, as described in section 5.4. In one embodiment, the chymotrypsin loop of the variant scaffold is a VEGF variant peptide e.g. SEQ ID NO:9, and the variant scaffold is altered further to comprise a combination of three amino acid substitutions at positions equivalent to positions 13, 50 and 52 of SEQ ID NO:187 to generate a modified variant BBPI scaffold. In one embodiment, the modified variant BBPI comprising a combination of two amino acid substitutions is the modified variant BBIt-AV-F50T-V52A of SEQ ID NO: 595 (DPDDESSKPCCDQCACTKSNPPQCRCSDMRLNSCHSACKSCACYNLYGWTCTCADITDFCYEPCKPSE; SEQ ID NO:595).

In some embodiments, the modified variant BBPI comprises a combination of three amino acid substitutions at amino acids at positions equivalent to positions 13, 50 and 52 of SEQ ID NO:187. In some embodiments, the combination of three amino acid substitutions is chosen from a combination of substitutions at positions 25-50-52, 29-50-52, 40-50-52, and 13-50-52. In some embodiments, the combination of three amino acid substitutions is chosen from 25L-50T-52A, 29P-50T-52A, 40K-50T-52A and 13I-50T-52A. The invention provides for any one of the variant BBPI scaffolds described in Section 5.3 and further comprising the combination of the three amino acid substitutions chosen from 25L-50T-52A, 29P-50T-52A, 40K-50T-52A and 13I-50T-52A, as described in section 5.4. In one embodiment, the chymotrypsin loop of the variant scaffold is a VEGF variant peptide e.g. SEQ ID NO:9, and the variant scaffold is altered further to comprise a combination of three amino acid substitutions at positions equivalent to positions 13, 50 and 52 of SEQ ID NO:187 to generate a modified variant BBPI scaffold.

In one embodiment, the modified variant BBPI comprising a combination of three amino acid substitutions is chosen from the modified variant BBIt-AV-S25L-F50T-V52A of SEQ ID NO: 603 (DPDDESSKPCCDQCACTKSNPPQCRCLDMRLNSCHSACKSCACYNLYGWTCTCADITDFCYEPCKPSE; SEQ ID NO:603), the modified variant BBIt-AV-L29P-F50T-V52A of SEQ ID NO:607 (DPDDESSKPCCDQCACTKSNPPQCRCSDMRPNSCHSACKSCACYNLYGWTCTCADITDFCYEPCKPSE; SEQ ID NO:607), and the modified variant BBIt-AV-A40K-F50T-V52A of SEQ ID NO:609 (DPDDESSKPCCDQCACTKSNPPQCRCSDMRLNSCHSACKSCKCYNLYGWTCTCADITDFCYEPCKPSE; SEQ ID NO:609).

In some embodiments, the modified variant BBPI comprises a combination of four amino acid substitutions at amino acids at positions equivalent to positions 13, 29, 50 and 52 of SEQ ID NO:187. In some embodiments, the combination of four amino acid substitutions is chosen from a combination of substitutions at positions 13-25-50-52, 13-29-50-52, 25-29-50-52, 13-40-50-52, 25-40-50-52, and 29-40-50-52. In some embodiments, the combination of four amino acid substitutions is chosen from 13I-25L-50T-52A, 13I-29P-50T-52A, 25L-29P-50T-52A, 13I-40K-50T-52A, 25L-40K-50T-52A, and 29P-40K-50T-52A. The invention provides for any one of the variant BBPI scaffolds described in Section 5.3 and further comprising the combination of the four amino acid substitutions chosen from 13I-25L-50T-52A, 13I-29P-50T-52A, 25L-29P-50T-52A, 13I-40K-50T-52A, 25L-40K-50T-52A, and 29P-40K-50T-52A, as described in section 5.4. In one embodiment, the chymotrypsin loop of the variant scaffold is a VEGF variant peptide chosen from SEQ ID NO:9 and 460, and the variant scaffold is altered further to comprise a combination of four amino acid substitutions at positions equivalent to positions 13, 29, 50 and 52 of SEQ ID NO:187 to generate a modified variant BBPI scaffold. In one embodiment, the modified variant BBPI comprising a combination of four amino acid substitutions is chosen from the modified variant BBIt-AV-A13I-S25L-F50T-V52A of SEQ ID NO:596 (DPDDESSKPCCDQCICTKSNPPQCRCLDMRLNSCHSACKSCACYNLYGWTCTCADITDFCYEPCKPSE; SEQ ID NO:596), the modified variant BBIt-AV-A13I-L29P-F50T-V52A of SEQ ID NO:600 (DPDDESSKPCCDQCICTKSNPPQCRCSDMRPNSCHSACKSCACYNLYGWTCTCADITDFCYEPCKPSE; SEQ ID NO:600), the modified variant BBIt-AV-A13I-A40K-F50T-V52A of SEQ ID NO:602 (DPDDESSKPCCDQCICTKSNPPQCRCSDMRLNSCHSACKSCKCYNLYGWTCTCADITDFCYEPCKPSE; SEQ ID NO:602), the modified variant BBIt-AV-S25L-L29P-F50T-V52A of SEQ ID NO:604 (DPDDESSKPCCDQCACTKSNPPQCRCLDMRPNSCHSACKSCACYNLYGWTCTCADITDFCYEPCKPSE; SEQ ID NO:604), the modified variant BBIt-AV-S25L-A40K-F50T-V52A of SEQ ID NO:606 (DPDDESSKPCCDQCACTKSNPPQCRCLDMRLNSCHSACKSCKCYNLYGWTCTCADITDFCYEPCKPSE; SEQ ID NO:606), the modified variant BBIt-AV-L29P-A40K-F50T-V52A of SEQ ID NO:608 (DPDDESSKPCCDQCACTKSNPPQCRCSDMRPNSCHSACKSCKCYNLYGWTCTCADITDFCYEPCKPSE; SEQ ID NO:608), and the modified variant BBIt-VEGKD-A13I-S25K-L29P-V52K of SEQ ID NO:643 (D P D D E S S K P C C D Q C I C T K S N P P Q C R C K D M R P N S C H S A C K S C I C K Y D L Y W W C F C K D I T D F C Y E P C K P S E; SEQ ID NO:643). In another embodiment, the chymotrypsin loop of the variant scaffold is an FGF5 variant peptide chosen from SEQ ID NOS:430 and 431, and the variant scaffold is altered further to comprise a combination of four amino acid substitutions at positions equivalent to positions 13, 29, 50 and 52 of SEQ ID NO:187 to generate a modified variant BBPI scaffold. In one embodiment, the modified variant BBPI comprising a combination of four amino acid substitutions is chosen from the modified variant BBIt-MM007-Q-A13I-L29P-F50T-V52A of SEQ ID NO:432, and the modified variant BBIt-FGFps2-Q-A13I-L29P-F50T-V52A of SEQ ID NO:434. In another embodiment, the chymotrypsin loop of the variant scaffold is a TGFβ variant peptide chosen from SEQ ID NOS:436, 437, 438, 672, 673, and 674 and the variant scaffold is altered further to comprise a combination of four amino acid substitutions at positions equivalent to positions 13, 29, 50 and 52 of SEQ ID NO:187 to generate a modified variant BBPI scaffold. In one embodiment, the modified variant BBPI comprising a combination of four amino acid substitutions is chosen from the modified variant BBIt-PEN3-Q-A13I-L29P-F50T-V52A of SEQ ID NO:443, the modified variant BBIt-MM021W-Q-A13I-L29P-F50T-V52A of SEQ ID NO:445, and the modified variant BBIt-WTQ-Q-A13I-L29P-F50T-V52A of SEQ ID NO:447.

In some embodiments, the modified variant BBPI comprises a combination of five amino acid substitutions at amino acids at positions equivalent to positions 13, 29, 40, 50 and 52 of SEQ ID NO:187. In some embodiments, the combination of five amino acid substitutions is chosen from a combination of substitutions at positions 13-25-29-50-52, 13-29-40-50-52, 13-25-40-50-52, 25-29-40-50-52, and 13-29-40-50-52. In some embodiments, the combination of five amino acid substitutions is chosen from 13I-25L-29P-50T-52A, 13I-29P-40K-50T-52A, 13I-25L-40K-50T-52A, 25L-29P-40K-50T-52A, 13L-29P-40K-50T-52A, 13I-29K-40K-50T-52A, 13I-29P-40K-50K-52A and 13I-29P-40K-50T-52T. The invention provides for any one of the variant BBPI scaffolds described in Section 5.3 and further comprising the combination of the five amino acid substitutions chosen from 13I-25L-29P-50T-52A, 13I-29P-40K-50T-52A, 13I-25L-40K-50T-52A, 25L-29P-40K-50T-52A, 13L-29P-40K-50T-52A, 13I-29K-40K-50T-52A, 13I-29P-40K-50K-52A and 13I-29P-40K-50T-52T, as described in section 5.4. In one embodiment, the chymotrypsin loop of the variant scaffold is a VEGF variant peptide of SEQ ID NO:9, and the variant scaffold is altered further to comprise a combination of five amino acid substitutions at positions equivalent to positions 13, 29, 40, 50 and 52 of SEQ ID NO:187 to generate a modified variant BBPI scaffold. In one embodiment, the modified variant BBPI comprising a combination of five amino acid substitutions is chosen from the modified variant BBIt-AV-A13I-S25L-L29P-F50T-V52A of SEQ ID NO:597 (DPDDESSKPCCDQCICTKSNPPQCRCLDMRPNSCHSACKSCACYNLYGWTCTCADITDFCYEPCKPSE; SEQ ID NO:597), the modified variant BBIt-AV-A13I-L29P-A40K-F50T-V52A of SEQ ID NO:599 (DPDDESSKPCCDQCICTKSNPPQCRCLDMRLNSCHSACKSCKCYNLYGWTCTCADITDFCYEPCKPSE; SEQ ID NO:599), the modified variant BBIt-AV-A13I-S25L-A40K-F50T-V52A of SEQ ID NO:601 (DPDDESSKPCCDQCICTKSNPPQCRCSDMRPNSCHSACKSCKCYNLYGWTCTCADITDFCYEPCKPSE; SEQ ID NO:601), the modified variant BBIt-AV-S25L-L29P-A40K-F50T-V52A of SEQ ID NO:605 (DPDDESSKPCCDQCACTKSNPPQCRCLDMRPNSCHSACKSCKCYNLYGWTCTCADITDFCYEPCKPSE; SEQ ID NO:605), the modified variant BBIt-AV-A13L-L29P-A40K-F50T-V52A of SEQ ID NO:615 (DPDDESSKPCCDQCLCTKSNPPQCRCSDMRPNSCHSACKSCKCYNLYGWTCTCADITDFCYEPCKPSE; SEQ ID NO:615), the modified variant BBIt-AV-A13I-L29K-A40K-F50T-V52A of SEQ ID NO:620 (DPDDESSKPCCDQCICTKSNPPQCRCSDMRKNSCHSACKSCKCYNLYGWTCTCADITDFCYEPCKPSE; SEQ ID NO:620), the modified variant BBIt-AV-A13I-L29P-A40K-F50K-V52A of SEQ ID NO:624 (DPDDESSKPCCDQCICTKSNPPQCRCSDMRPNSCHSACKSCKCYNLYGWTCKCADITDFCYEPCKPSE; SEQ ID NO:624), and the modified variant BBIt-AV-A13I-L29P-A40K-F50T-V52T of SEQ ID NO:625 (DPDDESSKPCCDQCICTKSNPPQCRCSDMRPNSCHSACKSCKCYNLYGWTCTCTDITDFCYEPCKPSE; SEQ ID NO:625).

In some embodiments, the modified variant BBPI comprises a combination of six amino acid substitutions at amino acids at positions equivalent to positions 13, 25, 29, 40, 50 and 52 of SEQ ID NO:187. In some embodiments, the combination of six amino acid substitutions is chosen from a combination of substitutions at positions 13-25-29-40-50-52, 1-13-29-40-50-52, 4-13-29-40-50-52, 5-13-29-40-50-52, 11-13-29-40-50-52, 13-25-29-40-50-52, 13-27-29-40-50-52, 13-29-31-40-50-52, 13-29-31-40-50-52, 13-29-38-40-50-52, and 13-29-38-40-50-52. In some embodiments, the combination of six amino acid substitutions is chosen from 13I-25L-29P-40K-50T-52A, 1C-13I-29P-40K-50T-52A, 4V-13I-29P-40K-50T-52A, 5P-13I-29P-40K-50T-52A, 11G-13I-29P-40K-50T-52A, 13I-25R-29P-40K-50T-52A, 13I-27R-29P-40K-50T-52A, 13I-29P-31A-40K-50T-52A, 13I-29P-31R-40K-50T-52A, 13I-29P-38N-40K-50T-52A, and 13I-29P-38N-40K-50T-52A. The invention provides for any one of the variant BBPI scaffolds described in Section 5.3 and further comprising the combination of the six amino acid substitutions chosen from 13I-25L-29P-40K-50T-52A, 1C-13I-29P-40K-50T-52A, 4V-13I-29P-40K-50T-52A, 5P-13I-29P-40K-50T-52A, 11G-13I-29P-40K-50T-52A, 13I-25R-29P-40K-50T-52A, 13I-27R-29P-40K-50T-52A, 13I-29P-31A-40K-50T-52A, 13I-29P-31R-40K-50T-52A, 13I-29P-38N-40K-50T-52A, and 13I-29P-38N-40K-50T-52A, as described in section 5.4. In one embodiment, the chymotrypsin loop of the variant scaffold is a VEGF variant peptide of SEQ ID NO:9, and the variant scaffold is altered further to comprise a combination of six amino acid substitutions at positions equivalent to positions 13, 25, 29, 40, 50 and 52 of SEQ ID NO:187 to generate a modified variant BBPI scaffold. In one embodiment, the modified variant BBPI comprising a combination of six amino acid substitutions is chosen from the modified variant BBIt-AV-A13I-S25L-L29P-A40K-F50T-V52A of SEQ ID NO:598 (DPDDESSKPCCDQCICTKSNPPQCRCLDMRPNSCHSACKSCKCYNLYGWTCTCADITDFCYEPCKPSE; SEQ ID NO:598), the modified variant BBIt-AV-D1C-A13I-L29P-A40K-F50T-V52A of SEQ ID NO:611 (DPDDEVSKPCCDQCICTKSNPPQCRCSDMRPNSCHSACKSCKCYNLYGWTCTCADITDFCYEPCKPSE; SEQ ID NO:611), the modified variant BBIt-AV-S4V-A13I-L29P-A40K-F50T-V52A of SEQ ID NO:612 (DPDDEVSKPCCDQCICTKSNPPQCRCSDMRPNSCHSACKSCKCYNLYGWTCTCADITDFCYEPCKPSE; SEQ ID NO:612), the modified variant BBIt-AV-S5P-A13I-L29P-A40K-F50T-V52A of SEQ ID NO:613 (DPDDESPKPCCDQCICTKSNPPQCRCSDMRPNSCHSACKSCKCYNLYGWTCTCADITDFCYEPCKPSE; SEQ ID NO:613), the modified variant BBIt-AV-Q11G-A13I-L29P-A40K-F50T-V52A of SEQ ID NO:614 (DPDDESSKPCCDGCICTKSNPPQCRCSDMRPNSCHSACKSCKCYNLYGWTCTCADITDFCYEPCKPSE; SEQ ID NO:614), the modified variant BBIt-AV-A13I-S25R-L29P-A40K-F50T-V52A- of SEQ ID NO:616 (DPDDESSKPCCDQCICTKSNPPQCRCRDMRPNSCHSACKSCKCYNLYGWTCTCADITDFCYEPCKPSE; SEQ ID NO:616), the modified variant BBIt-AV-A13I-M27R-L29P-A40K-F50T-V52A of SEQ ID NO:619 (DPDDESSKPCCDQCICTKSNPPQCRCSDRRPNSCHSACKSCKCYNLYGWTCTCADITDFCYEPCKPSE; SEQ ID NO:619), the modified variant BBIt-AV-A13I-L29P-S31A-A40K-F50T-V52A of SEQ ID NO:621 (DPDDESSKPCCDQCICTKSNPPQCRCSDMRPNACHSACKSCKCYNLYGWTCTCADITDFCYEPCKPSE; SEQ ID NO:621), the modified variant BBIt-AV-A13I-L29P-S31R-A40K-F50T-V52A of SEQ ID NO:622 (DPDDESSKPCCDQCICTKSNPPQCRCSDMRPNRCHSACKSCKCYNLYGWTCTCADITDFCYEPCKPSE; SEQ ID NO:622), the modified variant BBIt-AV-A13I-L29P-S38N-A40K-F50T-V52A of SEQ ID NO:623 (DPDDESSKPCCDQCICTKSNPPQCRCSDMRPNSCHSACKNCKCYNLYGWTCTCADITDFCYEPCKPSE; SEQ ID NO:623), and the modified variant BBIt-AV-A13I-L29P-S38N-A40K-F50T-V52A of SEQ ID NO:626 (DPDDESSKPCCDQCICTKSNPPQCRCSDMRPNSCHSACKSCKCYNLYGWTCTCADITDFCYEPCKPEE; SEQ ID NO:626).

In some embodiments, the modified variant BBPI comprises a combination of seven amino acid substitutions at amino acids at positions equivalent to positions 13, 25, 29, 31, 40, 50 and 52 of SEQ ID NO:187. In some embodiments, the combination of seven amino acid substitutions is chosen from a combination of substitutions at positions 13-25-29-31-40-50-52, 13-25-29-31-40-50-52, 13-25-27-29-31-50-52, 13-25-27-29-31-50-52, 13-25-27-29-31-50-52, 13-25-27-29-31-50-52, 13-25-27-29-31-50-52, 13-25-27-29-31-50-52, 13-25-27-29-31-50-52, 13-25-27-29-31-50-52, and 13-25-27-29-31-50-52. In some embodiments, the combination of seven amino acid substitutions is chosen from 13L-25R-29P-31A-40K-50T-52A, 13L-25R-29P-31R-40K-50T-52A, and 13I-25R-27A-29P-31A-50K-52T. The invention provides for any one of the variant BBPI scaffolds described in Section 5.3 and further comprising the combination of the six amino acid substitutions chosen from 13L-25R-29P-31A-40K-50T-52A, 13L-25R-29P-31R-40K-50T-52A, 13I-25R-27A-29P-31A-50K-52T, 13I-25R-27A-29P-31A-50K-52T, 13I-25R-27A-29P-31A-50K-52T, 13I-25R-27A-29P-31A-50K-52T, 13I-25R-27A-29P-31A-50K-52T, 13I-25R-27A-29P-31A-50K-52T, 13I-25R-27A-29P-31A-50K-52T, 13I-25R-27A-29P-31A-50K-52T, and 13I-25R-27A-29P-31A-50K-52T, as described in section 5.4. In one embodiment, the chymotrypsin loop of the variant scaffold is a VEGF variant peptide of SEQ ID NO:9, and the variant scaffold is altered further to comprise a combination of seven amino acid substitutions at positions equivalent to positions 13, 25, 29, 31, 40, 50 and 52 of SEQ ID NO:187 to generate a modified variant BBPI scaffold. In one embodiment, the modified variant BBPI comprising a combination of seven amino acid substitutions is chosen from the modified variant BBIt-AV-A13I-S25R-L29P-S31A-A40K-F50T-V52A of SEQ ID NO:617 (DPDDESSKPCCDQCICTKSNPPQCRCRDMRPNACHSACKSCKCYNLYGWTCTCADITDFCYEPCKPSE; SEQ ID NO:617), the modified variant BBIt-AV-A13I-S25R-L29P-S31R-A40K-F50T-V52A of SEQ ID NO:618 (DPDDESSKPCCDQCICTKSNPPQCRCRDMRPNRCHSACKSCKCYNLYGWTCTCADITDFCYEPCKPSE; SEQ ID NO:618), the modified variant of BBIt-VEGF-V1-A13I-S25R-M27A-L29P-S31A-F50K-V52T SEQ ID NO:491 (D P D D E S S K P C C D Q C I C T K S N P P Q C R C R D A R P N A C H S A C K S C A C S K H S Q I T C K C T D I T D F C Y E P C K P S E; SEQ ID NO:491), the modified variant BBIt-VEGF-V2-A13I-S25R-M27A-L29P-S31A-F50K-V52T of SEQ ID NO:632 (D P D D E S S K P C C D Q C I C T K S N P P Q C R C R D A R P N A C H S A C K S C A C K T N P S G S C K C T D I T D F C Y E P C K P S E: SEQ ID NO:632), the modified variant BBIt-VEGF-V3-A13I-S25R-M27A-L29P-S31A-F50K-V52T of SEQ ID NO:633 (D P D D E S S K P C C D Q C I C T K S N P P Q C R C R D A R P N A C H S A C K S C A C R P T G H S L C K C T D I T D F C Y E P C K P S E; SEQ ID NO:633), the modified variant BBIt-VEGF-V4-A13I-S25R-M27A-L29P-S31A-F50K-V52T of SEQ ID NO:634 (D P D D E S S K P C C D Q C I C T K S N P P Q C R C R D A R P N A C H S A C K S C A C K H S A K A E C K C T D I T D F C Y E P C K P S E; SEQ ID NO:634), the modified variant BBIt-VEGF-V5-A13I-S25R-M27A-L29P-S31A-F50K-V52T of SEQ ID NO:635 (D P D D E S S K P C C D Q C I C T K S N P P Q C R C R D A R P N A C H S A C K S C A C K P S S A S S C K C T D I T D F C Y E P C K P S E; SEQ ID NO:635), the modified variant BBIt-VEGF-V6-A13I-S25R-M27A-L29P-S31A-F50K-V52T of SEQ ID NO:636 (D P D D E S S K P C C D Q C I C T K S N P P Q C R C R D A R P N A C H S A C K S C A C P V T K R V H C K C T D I T D F C Y E P C K P S E; SEQ ID NO:636), the modified variant BBIt-TNFα-T1-A13I-S25R-M27A-L29P-S31A-F50K-V52T of SEQ ID NO:637 (D P D D E S S K P C C D Q C I C T K S N P P Q C R C R D A R P N A C H S A C K S C A C R Y W Q D I P C K C T D I T D F C Y E P C K P S E; SEQ ID NO:637), the modified variant BBIt-TNFα-T2-A13I-S25R-M27A-L29P-S31A-F50K-V52T of SEQ ID NO:638 (D P D D E S S K P C C D Q C I C T K S N P P Q C R C R D A R P N A C H S A C K S C A C A P E P I L A C K C T D I T D F C Y E P C K P S E; SEQ ID NO:638), and the modified variant BBIt-TNFα-T3-A13I-S25R-M27A-L29P-S31A-F50K-V52T of SEQ ID NO:639 (D P D D E S S K P C C D Q C I C T K S N P P Q C R C K D Q R P N E C H S A C K S C H C Y N L Y G W T C R C Q D I T D F C Y E P C K P P E; SEQ ID NO:639).

In some embodiments, the modified variant BBPI comprises a combination of eight amino acid substitutions at amino acids at positions equivalent to positions 13, 25, 27, 29, 31, 40, 50 and 52 of SEQ ID NO:187. In some embodiments, the combination of eight amino acid substitutions is chosen from a combination of substitutions at positions 13-25-27-29-31-40-50-52, 13-25-27-29-31-40-50-52, 13-25-27-29-31-40-50-52, 13-25-27-29-31-40-50-52, and 13-25-27-29-31-40-50-52. In some embodiments, the combination of eight amino acid substitutions is chosen from combinations 13I-25R-27A-29P-31A-40H-50K-52T, 13I-25K-27A-29R-31E-40K-50Q-52Q, 13I-25K-27R-29E-31A-40H-50R-52K, 13I-25K-27A-29R-31A-40H-50R-52L, and 13I-25K-27Q-29P-31E-40H-50R-52Q. The invention provides for any one of the variant BBPI scaffolds described in Section 5.3 and further comprising the combination of the eight amino acid substitutions chosen from combinations 13I-25R-27A-29P-31A-40H-50K-52T, 13I-25K-27A-29R-31E-40K-50Q-52Q, 13I-25K-27R-29E-31A-40H-50R-52K, 13I-25K-27A-29R-31A-40H-50R-52L, and 13I-25K-27Q-29P-31E-40H-50R-52Q, as described in section 5.4. In one embodiment, the chymotrypsin loop of the variant scaffold is a VEGF variant peptide of SEQ ID NO:9, and the variant scaffold is altered further to comprise a combination of seven amino acid substitutions at positions equivalent to positions 13, 25, 27, 29, 31, 40, 50 and 52 of SEQ ID NO:187 to generate a modified variant BBPI scaffold. In one embodiment, the modified variant BBPI comprising a combination of eight amino acid substitutions is chosen from the modified variant BBIt-AV-A13I-S25R-M27A-L29P-S31A-A40H-F50K-V52T of SEQ ID NO:627 (D P D D E S S K P C C D Q C I C T K S N P P Q C R C R D A R P N A C H S A C K S C H C Y N L Y G W T C K C T D I T D F C Y E P C K P S E; SEQ ID NO:627; KT8), the modified variant of BBIt-AV-A13I-S25K-M27A-L29R-S31E-A40K-F50Q-V52Q of SEQ ID NO:628 (D P D D E S S K P C C D Q C I C T K S N P P Q C R C K D A R R N E C H S A C K S C K C Y N L Y G W T C Q C Q D I T D F C Y E P C K P S E; SEQ ID NO:628; QQ8), the modified variant BBIt-AV-A13I-S25K-M27R-L29E-S31A-A40H-F50R-V52K of SEQ ID NO:629 (D P D D E S S K P C C D Q C I C T K S N P P Q C R C K D R R E N A C H S A C K S C H C Y N L Y G W T C R C K D I T D F C Y E P C K P S E; SEQ ID NO:629; RK8), the modified variant BBIt-AV-A13I-S25K-M27A-L29R-S31A-A40H-F50R-V52L of SEQ ID NO:630 (D P D D E S S K P C C D Q C I C T K S N P P Q C R C K D A R R N A C H S A C K S C H C Y N L Y G W T C R C L D I T D F C Y E P C K P S E; SEQ ID NO: 630; RL8) and the modified variant BBIt-AV-A13I-S25K-M27Q-L29P-S31E-A40H-F50R-V52Q of SEQ ID NO:631 (D P D D E S S K P C C D Q C I C T K S N P P Q C R C K D Q R P N E C H S A C K S C H C Y N L Y G W T C R C Q D I T D F C Y E P C K P S E; SEQ ID NO:631; RQ8).

The invention further provides for modified variant BBPIs that comprise any one combination of the amino acid substitutions described above and that have greater protease inhibitory activity than the unmodified precursor variant BBPI. In some embodiments, modified variant BBPIs which contain a variant peptide in place of the chymotrypsin loop of the corresponding precursor unmodified BBPI have greater trypsin inhibitory activity (TIA) than that of the precursor unmodified BBPI scaffold. In other embodiments, modified variant BBPIs which contain a variant peptide in place of the trypsin loop of the corresponding precursor unmodified BBPI have greater chymotrypsin inhibitory activity (TIA) than that of the precursor unmodified BBPI scaffold.

As shown in the Examples, substitutions of at least one amino acid in the backbone of the variant BBPI generates a modified variant BBPI that has a greater production yield that the unmodified variant BBPI. In some embodiments, BBPIs that comprise a combination of two, three, four, five, six, seven or eight amino acid substitutions have a greater production yield than the unmodified precursor BBPI. Thus, the invention provides for modified variant BBPIs that comprise any one combination of the amino acid substitutions described above and that have greater production yield (PY) than the unmodified precursor variant BBPIs. In yet other embodiments, the invention provides for modified variant BBPIs that comprise any one combination of the amino acid substitutions described above and that have greater trypsin inhibitory activity and greater production yield than the TIA and PY of the unmodified precursor variant BBPIs.

In some embodiments, the modified variant BBPIs further comprise a peptide insert that is positioned at the N-terminus of the modified variant BBPI. In some embodiments, the peptide insert comprises a sequence of between 1 and 15 amino acids. In other embodiments, the peptide insert comprises a sequence between 5 and 10 amino acids. In some embodiments, the peptide insert comprises the peptide of SEQ ID NO:389 (DDEPSKPCCDPDP; SEQ ID NO:389). Examples of modified variant BBPIs that the peptide insert of SEQ ID NO:389 are the modified variant 4D13BBIt-AV of (DDEPSKPCCDPDPDDESSKPCCDQCACTKSNPPQCRCSDMRLNSCHSACKSCACYNLYGWTCFCVDITDFCYEPCKPSE; SEQ ID NO:390), and the modified variant BBIt-AV-4D13-13I-29P-40K-50T-52A of SEQ ID NO: 413 (DPDDEPSKPCCDPDPDDESSKPCCDQCICTKSNPPQCRCSDMRPNSCHSACKSCKCYNLYGWTCTCADITDFCYEPCKPSE; SEQ ID NO:413).

5.4.3 BBPI Fusion Proteins

In some embodiments, each modified variant BBPI is expressed as fusion protein comprising a catalytic domain, a cleavage site and the BBPI scaffold. The catalytic domain is chosen from cellulase, cutinase, and disulfide isomerase. In some embodiments, the catalytic domain comprised in the BBPI fusion protein is the cellulase catalytic domain of SEQ ID NO:669

(SEQ ID NO:669) DDYSVVEEHGQLSISNGELVNERGEQVQLKGMSSHGLQWYGQFVNYESMK WLRDDWGITVFRAAMYTSSGGYIDDPSVKEKVKETVEAAIDLGIYVIIDW HILSDNDPNIYKEEAKDFFDEMSELYGDYPNVIYEIANEPNGSDVTWDNQ IKPYAEEVIPVIRDNDPNNIVIVGTGTWSQDVHHAADNQLADPNVMYAFH FYAGTHGQNLRDQVDYALGQGAAIFVSEWGTSAATGDGGVFLDEAQVWID FMDERNLSWANWSLTHKDESSAALMPGANPTGGWTEAELSPSGTFVREKI RESAS

The fusion protein is processed by a protease or acid/heat treatment to liberate the modified variant BBPI. In some embodiments, the fusion protein further comprises at least one linker sequence. In some embodiments, the linker sequence is selected from the group consisting of SEQ ID NOS:141-143. Although cleavage of the fusion polypeptide to release the modified variant BBPI will often be useful, it is not necessary. Modified variant BBPIs expressed and secreted as fusion proteins surprisingly retain their function.

The modified variant BBPI fusion proteins are each expressed by the host bacterial cell from a fusion polynucleotide sequence. Such fusion polynucleotide sequences are assembled in proper reading frame from the 5′ terminus to 3′ terminus in the order of first, second, third and fourth polynucleotide sequences. As so assembled, the polynucleotide sequence encodes a “fusion polypeptide” encoding from its amino-terminus 1. a signal peptide functional as a secretory sequence in a bacterial species, 2. a secreted polypeptide or portion thereof normally secreted from a bacterial species e.g. cellulase or portion thereof, 3. a cleavable linker peptide and a 4. desired polypeptide (e.g., a modified variant BBPI). In some embodiments, the above-defined fusion polynucleotide sequence further comprises a polynucleotide encoding a portion of a propeptide that functions as a spacer between the first and second polynucleotide sequences of a fusion protein. The function of the spacer is intended to increase the distance between the first and second encoded polypeptides. In some embodiments, the spacer sequence is 1-10 amino acids long. In other embodiments, the above-defined fusion polynucleotide sequence further comprises a polynucleotide encoding a peptide insert between the linker peptide and the modified variant BBPI. In some embodiments, the peptide insert comprises a sequence of between 1 and 15 amino acids. In other embodiments, the peptide insert comprises a sequence between 5 and 10 amino acids. In some embodiments, the peptide insert comprises the peptide of SEQ ID NO:389.

Various methods are known to those in the art for the production of fusion proteins (See e.g., U.S. Pat. Nos. 5,411,873, 5,429,950, and 5,679,543, all of which are incorporated by reference herein). Thus, it is intended that any suitable method will find use in the present invention.

5.5 BOWMAN BIRK PROTEASE INHIBITOR POLYNUCLEOTIDES

To the extent that the present invention depends on the production of fusion proteins, it relies on routine techniques in the field of recombinant genetics. Basic texts disclosing the general methods of use in this invention include Sambrook et al., Molecular Cloning, A Laboratory Manual ((2nd ed.) [1989]); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Ausubel et al., (eds.), Current Protocols in Molecular Biology (1994).

The invention provides for compositions including polynucleotide constructs, vectors and host cells that enable the expression of modified variant BBPIs. The polynucleotide constructs of the invention comprise a promoter sequence and a fusion polynucleotide sequence that encodes a fusion protein comprising a modified variant BBPI. As described above, the fusion polynucleotide sequence comprises a catalytic domain, a cleavage site and the BBPI scaffold. Natural or synthetic polynucleotide fragments encoding a BBPI may be incorporated into the polynucleotide constructs. The at least one amino acid substitution introduced into a variant BBPI is generated by means of site saturation mutagenesis in at least one codon. In alternative embodiments, the at least one amino acid substitution is encoded by DNA oligonucleotides that contain the encoding sequence, and that are annealed and ligated into the protease inhibitor DNA sequence. The desired DNA sequence is then isolated and used in the methods provided herein.

In some embodiments, the polynucleotide constructs of the invention comprise polynucleotide sequences that encode a modified variant BBPI that shares at least about 65% amino acid sequence identity, at least about 70% amino acid sequence identity, at least about 75% amino acid sequence identity, at least about 80% amino acid sequence identity, at least about 85% amino acid sequence identity, at least about 90% amino acid sequence identity, at least about 92% amino acid sequence identity, at least about 95% amino acid sequence identity, at least about 97% amino acid sequence identity, at least about 98% amino acid sequence identity, and at least about 99% amino acid sequence identity with the amino acid sequence of the unmodified precursor variant BBPI and has greater protease inhibitory activity than the unmodified precursor variant BBPI. The invention further provides for polynucleotides encoding modified variant BBPIs that comprise any one combination of the amino acid substitutions described above and that have greater protease inhibitory activity e.g. trypsin inhibitory activity, than the unmodified precursor variant BBPI.

In some embodiments, the polynucleotide constructs of the invention comprise a polynucleotide sequence that may be codon optimized for expression of a modified variant BBPI in the host cell used. Since codon usage tables listing the usage of each codon in many cells are known in the art (See, e.g., Nakamura et al., Nucl. Acids Res., 28:292 [2000]) or readily derivable, such nucleic acids can be readily designed giving the amino acid sequence of a protein to be expressed.

The invention also encompasses polynucleotide constructs that comprise coding sequences encoding modified variant BBPI proteins that are related by being structurally and/or functionally similar. In some embodiments, a modified variant BBPI is derived from a naturally-occurring BBPI belonging to a different genus and/or species. In some embodiments, related proteins are provided from the same species. Indeed, it is not intended that the present invention be limited to related proteins from any particular source(s). In addition, the term “related proteins” encompasses tertiary structural homologs and primary sequence homologs. For example, the present invention encompasses such homologues including but not limited to such BBPI proteins such as those described by Prakash et al. (J mol Evol 42:560-569 [1996]).

In some embodiments, the promoter sequence comprised in the polynucleotide constructs of the invention is operably linked to the BBPI-encoding polynucleotide. Exemplary promoters include both constitutive promoters and inducible promoters. Such promoters are well known to those of skill in the art. Those skilled in the art are also aware that a natural promoter can be modified by replacement, substitution, addition or elimination of one or more nucleotides without changing its function. The practice of the present invention encompasses and is not constrained by such alterations to the promoter. The choice of promoter used in the genetic construct is within the knowledge of one skilled in the art.

In some embodiments, the promoter sequence may be obtained from a bacterial source. In some embodiments, the promoter sequence may be obtained from a Gram-positive bacterium such as a Bacillus strain (e.g., Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, or Bacillus thuringiensis); or a Streptomyces strain (e.g., Streptomyces lividans or Streptomyces murinus); or from a gram negative bacterium (e.g., E. coli or Pseudomonas sp.).

The promoter can be any DNA sequence having transcription activity in the host organism of choice and can be derived from genes that are homologous or heterologous to the host organism. Examples of suitable promoters that can be used to express a modified variant BBPI in a bacterial host include, but are not limited to the promoter of the lac operon of E. coli, the Streptomyces coelicolor agarase gene dagA promoters, the promoters of the Bacillus licheniformis α-amylase gene (amyL), the aprE promoter of Bacillus subtilis, the promoters of the Bacillus stearothermophilus maltogenic amylase gene (amyM), the promoters of the Bacillus amyloliquefaciens α-amylase gene (amyQ), the promoters of the Bacillus subtilis xylA and xylB genes and a promoter derived from a Lactococcus sp.-derived promoter including the PI70 promoter. When the gene encoding the compound is expressed in a bacterial species such as E. coli, a suitable promoter can be selected, for example, from a bacteriophage promoter including a T7 promoter and a phage lambda promoter. For transcription in a fungal species, examples of useful promoters are those derived from the genes encoding the Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, A. niger neutral α-amylase, A. niger acid stable α-amylase, A. niger glucoamylase, Rhizomucor miehei lipase, A. oryzae alkaline protease, A. oryzae triose phosphate isomerase, and A. nidulans acetamidase. Examples of suitable promoters for the expression in a yeast species include but are not limited to the Gal 1 and Gal 10 promoters of Saccharomyces cerevisiae and the Pichia pastoris AOX1 or AOX2 promoters.

The invention also encompasses promoter sequences that have been mutated to increase the activity of the promoter when compared to the activity of the corresponding wild-type promoter resulting in the expression of the modified variant BBPI protein. Thus, it is understood that variants of the sequences that define the B. subtilis AprE promoter find use in the constructs of the invention. Methods for creating promoter variants in Bacillus sp. are well known in the art (See e.g., Helmann et al., 2002. RNA polymerase and sigma factors, pp 289-312 In A. L. Sonenshein, J. A. Hoch and R. Losick (ed), Bacillus subtilis and its closest relatives: from genes to cells. American Society for Microbiology, Washington, D.C.) It is not intended that the present invention be limited to any particular promoter, as any suitable promoter known to those skilled in the art finds use with the present invention.

In embodiments, in addition to a promoter sequence, the polynucleotide construct also contains a transcription termination region downstream of the structural gene to provide for efficient termination. In some embodiments, the termination region is obtained from the same gene as the promoter sequence, while in other embodiments it is obtained from another gene. The selection of suitable transcription termination signals is well-known to those of skill in the art.

5.6 BOWMAN BIRK PROTEASE INHIBITOR VECTORS

The invention provides vectors comprising the polynucleotide constructs of the invention. The vectors are introduced into a host cell to express the modified variant BBPI proteins of the invention. Any vector may be used as long as it is replicable and viable in the cells into which it is introduced. Large numbers of suitable vectors and promoters are known to those of skill in the art, and are commercially available. Appropriate cloning and expression vectors are also described in various references known to those in the art (See e.g., Sambrook et al., supra and Ausubel et al., supra, expressly incorporated by reference herein). The appropriate BBPI-encoding DNA sequence is inserted into a plasmid or vector (collectively referred to herein as “vectors”) by any suitable method. In general, the DNA sequence is inserted into an appropriate restriction endonuclease site(s) by standard procedures known to those in the art.

Appropriate vectors are typically equipped with a selectable marker-encoding nucleic acid sequence, insertion sites, and suitable control elements, such as termination sequences. In some embodiments, the vectors comprise regulatory sequences, including, for example, control elements (i.e., promoter and terminator elements or 5′ and/or 3′ untranslated regions), effective for expression of the coding sequence in host cells (and/or in a vector or host cell environment in which a modified soluble protein coding sequence is not normally expressed), operably linked to the coding sequence. Large numbers of suitable vectors and promoters are known to those of skill in the art, many of which are commercially available and known to those in the art. The choice of the proper selectable marker will depend on the host cell. Appropriate markers for different bacterial hosts are well known in the art. Typical selectable marker genes encode proteins that (a) confer resistance to antibiotics or other toxins (e.g., ampicillin, methotrexate, tetracycline, neomycin mycophenolic acid, puromycin, zeomycin, or hygromycin; or (b) complement an auxotrophic mutation or a naturally occurring nutritional deficiency in the host strain.

In some embodiments, expression of a fusion BBPI polypeptide results from the expression of one or more copies of the corresponding fusion polypeptide-encoding polynucleotide that is present on a multicopy/replicating plasmid that has been introduced into a host cell. In some embodiments, the vector is a multicopy/replicating plasmid vector which forms an extrachromosomal self-replicating genetic element that expresses a fusion BBPI protein in the host cell. Typically, the vector is a plasmid vector, which carries a selectable marker gene that allows for ease of selecting the host cells that contain the plasmid. Vectors that replicate autonomously in a host cell include vectors that comprise an origin of replication, which enables the vector to replicate autonomously in the Bacillus cell. Examples of bacterial origins of replication are the origins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permitting replication in E. coli, and pUB110, pC194, pE194, pTA1060, and pAMβ1 permitting replication in Bacillus. The origin of replication may be one having a mutation to make its function temperature-sensitive in the Bacillus cell (See, e.g., Ehrlich, Proceedings of the National Academy of Sciences USA 75:1433 [1978]). Additional bacterial expression vectors that find use in the present invention include bacteriophages λ and M13, and fusion expression systems such as MBP, GST, and LaZ. In further embodiments, epitope tags are added to recombinant proteins, in order to provide convenient methods of isolation (e.g., c-myc).

In some embodiments, the expression of a BBPI fusion polypeptide results from the expression of at least one copy of a BBPI fusion-encoding polynucleotide that is integrated into the genome of the host cell. Thus, in some embodiments, the invention provides a BBPI encoding polynucleotide construct that is incorporated into an integrating vector. Thus, when the vector is introduced into the host cell, it is integrated into the genome and replicated together with the genome into which it has integrated. Multiple copies of the BBPI gene can be integrated at several positions in the genome of the host cell. Alternatively, an amplifiable expression cassette carrying a sequence encoding a BBPI fusion protein and a selectable marker (e.g., an antimicrobial resistance marker, such as a gene coding chloramphenicol acetyl transferase) can be integrated in the genome via a single cross-over event and then amplified by challenging the transformed host cell with increasing concentrations of the appropriate antimicrobial (e.g., chloramphenicol).

5.7 BOWMAN BIRK PROTEASE INHIBITOR HOST CELLS

In one embodiment, the invention provides for a host cell transformed with an expression vector comprising a polynucleotide sequence encoding a modified variant BBPI. After the expression vector is introduced into the host cells, the transformed host cells are cultured under conditions favoring expression of gene encoding the desired BBPI fusion protein. Large batches of transformed cells can be cultured as described above. Finally, product is recovered from the culture using techniques known in the art.

Methods for introducing DNA into Bacillus cells involving plasmid constructs and transformation of plasmids into bacterial host cells are well known. In some embodiments, the plasmids are subsequently isolated from E. coli and transformed into Bacillus. However, it is not essential to use intervening microorganisms such as E. coli, and in some embodiments, a DNA construct or vector is directly introduced into a Bacillus host. Those of skill in the art are well aware of suitable methods for introducing polynucleotide sequences into Bacillus cells (See e.g., Ferrari et al., “Genetics,” in Harwood et al. (ed.), Bacillus, Plenum Publishing Corp. [1989], pages 57-72; Saunders et al., J. Bacteriol, 157:718-726 [1984]; Hoch et al., J. Bacteriol., 93:1925-1937 [1967]; Mann et al., Current Microbiol., 13:131-135 [1986]; and Holubova, Folia Microbiol., 30:97 [1985]; Chang et al., Mol. Gen. Genet., 168:11-115 [1979]; Vorobjeva et al., FEMS Microbiol. Lett., 7:261-263 [1980]; Smith et al., Appl. Env. Microbiol., 51:634 [1986]; Fisher et al., Arch. Microbiol., 139:213-217 [1981]; and McDonald, J. Gen. Microbiol., 130:203 [1984]). Indeed, such methods as transformation, including protoplast transformation and congression, transduction, and protoplast fusion are known and suited for use in the present invention. Methods of transformation are particularly preferred to introduce a DNA construct provided by the present invention into a host cell.

In addition to commonly used methods, in some embodiments, host cells are directly transformed (i.e., an intermediate cell is not used to amplify, or otherwise process, the DNA construct prior to introduction into the host cell). Introduction of the DNA construct into the host cell includes those physical and chemical methods known in the art to introduce DNA into a host cell without insertion into a plasmid or vector. Such methods include, but are not limited to electroporation, insertion of naked DNA or liposomes and the like. In additional embodiments, DNA constructs are co-transformed with a plasmid, without being inserted into the plasmid. In further embodiments, a selective marker is deleted from the altered Bacillus strain by methods known in the art (See, Stahl et al., J. Bacteriol., 158:411-418 [1984]; and Palmeros et al., Gene 247:255-264 [2000]).

Methods known in the art to transform Bacillus, include such methods as plasmid marker rescue transformation, which involves the uptake of a donor plasmid by competent cells carrying a partially homologous resident plasmid (Contente et al., Plasmid 2:555-571 [1979]; Haima et al., Mol. Gen. Genet., 223:185-191 [1990]; Weinrauch et al., J. Bacteriol., 154:1077-1087 [1983]; and Weinrauch et al., J. Bacteriol., 169:1205-1211 [1987]). In this method, the incoming donor plasmid recombines with the homologous region of the resident “helper” plasmid in a process that mimics chromosomal transformation.

Other methods involving transformation by protoplast transformation are well known in the art (See e.g., Chang and Cohen, Mol. Gen. Genet., 168:111-115 [1979]; Vorobjeva et al., FEMS Microbiol. Lett., 7:261-263 [1980]; Smith et al., Appl. Env. Microbiol., 51:634 [1986]; Fisher et al., Arch. Microbiol., 139:213-217 [1981]; McDonald [1984] J. Gen. Microbiol., 130:203 [1984]; and Bakhiet et al., 49:577 [1985]). In addition, Mann et al., (Mann et al., Curr. Microbiol., 13:131-135 [1986]) describe transformation of Bacillus protoplasts, and Holubova (Holubova, Microbiol., 30:97 [1985]) describe methods for introducing DNA into protoplasts using DNA-containing liposomes. In some embodiments, marker genes are used in order to indicate whether or not the gene of interest is present in the host cell. In some embodiments, the BBPI fusion polynucleotide sequence contained in the vector of the invention encodes for a BBPI fusion protein having SEQ ID NO:195. In addition to these methods, in other embodiments, host cells are directly transformed. In “direct transformation,” an intermediate cell is not used to amplify, or otherwise process, the modified polynucleotide prior to introduction into the host (i.e., Bacillus) cell. Introduction of the modified polynucleotide into the host cell includes those physical and chemical methods known in the art to introduce modified polynucleotide into a host cell without insertion into a plasmid or vector. Such methods include but are not limited to the use of competent cells, as well as the use of “artificial means” such as calcium chloride precipitation, electroporation, etc. to introduce DNA into cells. Thus, the present invention finds use with naked DNA, liposomes and the like.

Examples of suitable bacterial host organisms are Gram positive species, including, but not limited to members of the Bacillus species, which Bacillaceae, (e.g., B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. clausii, B. halodurans, B. coagulans, B. circulans, B. lautus, B. megaterium and B. thuringiensis), Streptomyces species (e.g., S. murinus and S. lividans) lactic acid bacteria (e.g., Lactococcus spp. such as Lactococcus lactis; Lactobacillus spp. including Lactobacillus reuteri; Leuconostoc spp.; Pediococcus spp.; and Streptococcus spp. Alternatively, strains of Gram-negative species belonging to Enterobacteriaceae (e.g., E. coli) or members of the Pseudomonadaceae find use in the present invention.

In some embodiments, a suitable yeast host organism is selected from various biotechnologically useful yeasts species, including but not limited to Pichia sp., Hansenula sp or Kluyveromyces, Yarrowinia, Saccharomyces (e.g., Saccharomyces cerevisiae), Schizosaccharomyce (e.g., S. pombe). In some embodiments, strains of the methylotrophic yeast species Pichia pastoris are used as the host organism, while in other embodiments, the host organism is a Hansenula species. Suitable host organisms among filamentous fungi include species of Aspergillus (e.g., A. niger, A. oryzae, A. tubigensis, A. awamori and Aspergillus nidulans). Alternatively, strains of Fusarium species (e.g. F. oxysporum) and Rhizomucor (e.g., Rhizomucor miehei) find used as the host organism. Additional suitable strains include, but are not limited to Thermomyces and Mucor species.

Accessory proteins such as thiol-disulfide oxidoreductases or chaperones find use in some embodiments, as they may be beneficial to help fold the secretory protein into its active conformation. Thiol-disulfide oxidoreductases and protein disulfide isomerases catalyze the formation of the correct disulfide bonds in the protein. Overexpression of the bdbDC operon in B. subtilis has been shown to be beneficial for the production of a protein with disulfide bonds (See e.g., Meima et al., J. Biol. Chem., 277:6994-7001, [2002]). Chaperones help the secretory protein to fold by binding to exposed hydrophobic regions in the unfolded states and preventing unfavourable interactions and prolyl-peptidyl cis-trans isomerases assist in formation of the proper conformation of the peptide chain adjacent to proline residues.

In some embodiments of the present invention, the host cells are transformed with an expression vector encoding at least one thiol-disulfide oxidoreductase or chaperone. It is not intended that the present invention be limited to any particular thiol-disulfide oxidoreductase or chaperone, as any suitable thiol-disulfide oxidoreductase or chaperone known to those skilled in the art will find use in the present invention.

In some embodiments of the present invention, and as described further below, the fraction of properly folded secretory protein is increased by the addition of chemicals to the growth medium that reduce/oxidize disulfide bonds, and/or alter the general redox potential, and/or chemicals that alter solvent properties thus affecting protein conformation and aggregation. In particularly preferred embodiments, a reagent that reduces disulfide bonds, such as 2-mercaptoethanol (βME), is preferred to increase the fraction of correctly folded protein. However, in other embodiments and depending on the medium used, other disulfide reducing or oxidizing agents (e.g., DTT, TCEP, reduced and oxidized glutathione, cysteine, cystine, cysteamine, thioglycolate, S₂O₃ ²⁻, S₂O₄ ²⁻, S₂O₅ ²⁻, SO₃ ²⁻, S₂O₇ ²⁻, Cu+, etc.), either used alone or in combination, find use in the present invention. It is contemplated that other adjuvants that alter solvent properties, (e.g., urea, DMSO, TWEEN®-80, etc.), either added to the growth medium alone or preferably in combination with disulfide reducing/oxidizing agents, such as □ME, will also increase the fraction of correctly folded secretory protein and find use in various embodiments of the present invention. In some preferred embodiments, the BME is used at concentrations ranging from 0.5 to 4 mM, while in other embodiments, the concentrations range from 0.1 mM to 10 mM. Indeed, those of skill in the art know how to select the best growth medium and growth conditions to optimize the effects of the added thiol reducing/oxidizing agents and/or other adjuvants, as well as the concentration of thio reducing/oxidizing agents and/or other adjuvants to use. It is not intended that the present invention be limited to any particular disulfide reducing/oxidizing agent or adjuvant, as any suitable reagents known to those skilled in the art find use in the present invention.

5.7.1 Fermentation Parameters

The present invention relies on fermentation procedures for culturing bacterial species. Fermentation procedures for production of heterologous proteins by bacterial species are well known in the art. Culturing is accomplished in a growth medium comprising an aqueous mineral salts medium, organic growth factors, the carbon and energy source material, molecular oxygen (for aerobic and facultative bacteria), and, of course, a starting inoculum of one or more particular microorganism species to be employed.

In addition to the carbon and energy source, oxygen, assimilable nitrogen, and an inoculum of the microorganism, it is necessary to supply suitable amounts in proper proportions of mineral nutrients to assure proper microorganism growth, maximize the assimilation of the carbon and energy source by the cells in the microbial conversion process, and achieve maximum cellular yields with maximum cell density in the fermentation medium.

Various culture media find use in the present invention, as known to those of skill in the art. However, standard bacterial culture media find use in the present invention. In some preferred media formulations, the media include, in addition to nitrogen, suitable amounts of phosphorus, magnesium, calcium, potassium, sulfur, and sodium, in suitable soluble assimilable ionic and combined forms, and also present preferably should be certain trace elements such as copper, manganese, molybdenum, zinc, iron, boron, and iodine, and others, again in suitable soluble assimilable form, all as known in the art.

In some embodiments, the fermentation reaction involves an aerobic process in which the molecular oxygen needed is supplied by a molecular oxygen-containing gas such as air, oxygen-enriched air, or even substantially pure molecular oxygen, provided to maintain the contents of the fermentation vessel with a suitable oxygen partial pressure effective in assisting the microorganism species to grow in a thriving fashion. In effect, by using an oxygenated hydrocarbon substrate, the oxygen requirement for growth of the microorganism is reduced. Nevertheless, molecular oxygen must be supplied for growth of aerobic and to a lesser extent, facultative organisms.

Although the aeration rate can vary over a considerable range, aeration generally is conducted at a rate which is in the range of about 0.5 to 10, preferably about 0.5 to 7, volumes (at the pressure employed and at 25° C.) of oxygen-containing gas per liquid volume in the fermentor per minute. This amount is based on air of normal oxygen content being supplied to the reactor, and in terms of pure oxygen the respective ranges would be about 0.1 to 1.7, or preferably about 0.1 to 1.3, volumes (at the pressure employed and at 25° C.) of oxygen per liquid volume in the fermentor per minute.

The pressure employed for the microbial conversion process can range widely. Pressures generally are within the range of about 0 to 50 psig, presently preferably about 0 to 30 psig, more preferably at least slightly over atmospheric pressure, as a balance of equipment and operating cost versus oxygen solubility achieved. Greater than atmospheric pressures are advantageous in that such pressures do tend to increase a dissolved oxygen concentration in the aqueous ferment, which in turn can help increase cellular growth rates. At the same time, this is balanced by the fact that high atmospheric pressures do increase equipment and operating costs.

The fermentation temperature can vary somewhat, but for most bacterial species used in the present invention, the temperature generally will be within the range of about 20° C. to 40° C., generally preferably in the range of about 28° C. to 37° C., depending on the strain of microorganism chosen, as known to those skilled in the art.

The microorganisms also require a source of assimilable nitrogen. The source of assimilable nitrogen can be any nitrogen-containing compound or compounds capable of releasing nitrogen in a form suitable for metabolic utilization by the microorganism. While a variety of organic nitrogen source compounds, such as protein hydrolysates, can be employed, usually cheap nitrogen-containing compounds such as ammonia, ammonium hydroxide, urea, and various ammonium salts such as ammonium phosphate, ammonium sulfate, ammonium pyrophosphate, ammonium chloride, or various other ammonium compounds can be utilized. Ammonia gas itself is convenient for large scale operations, and can be employed by bubbling through the aqueous ferment (fermentation medium) in suitable amounts. At the same time, such ammonia can also be employed to assist in pH control.

The pH range in the aqueous microbial ferment (fermentation admixture) should be in the exemplary range of about 2.0 to 8.0. However, pH range optima for certain microorganisms are dependent on the media employed to some extent, as well as the particular microorganism, and thus change somewhat with change in media as known to those skilled in the art.

While the average retention time of the fermentation admixture in the fermentor can vary considerably, depending in part on the fermentation temperature and culture employed, as known in the art.

In some embodiments, the fermentation is preferably conducted in such a manner that the carbon-containing substrate can be controlled as a limiting factor, thereby providing good conversion of the carbon-containing substrate to cells and avoiding contamination of the cells with a substantial amount of unconverted substrate. The latter is not a problem with water-soluble substrates, since any remaining traces are readily removed. It may be a problem, however, in the case of non-water-soluble substrates, and require added product-treatment steps such as suitable washing steps. The time needed to reach this limiting substrate level is not critical and may vary with the particular microorganism and fermentation process being conducted. However, it is well known in the art how to determine the carbon source concentration in the fermentation medium and whether or not the desired level of carbon source has been achieved.

Although in some embodiments, the fermentation is conducted as a batch or continuous operation, fed batch operation is generally preferred for ease of control, production of uniform quantities of products, and most economical uses of all equipment.

If desired, part or all of the carbon and energy source material and/or part of the assimilable nitrogen source such as ammonia can be added to the aqueous mineral medium prior to feeding the aqueous mineral medium into the fermentor. Indeed, each of the streams introduced into the reactor preferably is controlled at a predetermined rate, or in response to a need determinable by monitoring such as concentration of the carbon and energy substrate, pH, dissolved oxygen, oxygen or carbon dioxide in the off-gases from the fermentor, cell density measurable by light transmittancy, or the like. The feed rates of the various materials can be varied so as to obtain as rapid a cell growth rate as possible, consistent with efficient utilization of the carbon and energy source, to obtain as high a yield of microorganism cells relative to substrate charge as possible, but more importantly to obtain the highest production of the desired protein per unit volume.

In either a batch, or the preferred fed batch operation, all equipment, reactor, or fermentation means, vessel or container, piping, attendant circulating or cooling devices, and the like, are initially sterilized, usually by employing steam such as at about 121° C. for at least about 15 minutes. The sterilized reactor then is inoculated with a culture of the selected microorganism in the presence of all the required nutrients, including oxygen, and the carbon-containing substrate. The type of fermentor employed is not critical, though in some embodiments, the 15L Biolafitte (Saint-Germain-en-Laye, France) is preferred.

5.8 PROTEIN SEPARATION

In some embodiments, host cells transformed with polynucleotide sequences encoding modified proteases are cultured under conditions suitable for the expression and recovery of the encoded protein from cell culture. The protein produced by a recombinant host cell comprising a fusion BBPI of the present invention is secreted into the culture media. In some embodiments, the secreted fusion BBPI is recovered.

In some embodiments, the present invention provides methods of separating a desired protein from its fusion analog. It is contemplated that the methods described herein will find use in the separation of the modified variant BBPI from the fusion analog.

The collection and purification of the desired fusion BBPI protein from the fermentation broth can also be achieved using procedures known to those of skill in the art. The fermentation broth will generally contain cellular debris, including cells, various suspended solids and other biomass contaminants, as well as the desired protein product, which are preferably removed from the fermentation broth by means known in the art. Suitable processes for such removal include conventional solid-liquid separation techniques (e.g., centrifugation, filtration, dialysis, microfiltration, rotary vacuum filtration, or other known processes), to produce a cell-free filtrate. In some embodiments, it is preferable to further concentrate the fermentation broth or the cell-free filtrate prior to the purification and/or crystallization process using techniques such as ultrafiltration, evaporation and/or precipitation.

Precipitating the proteinaceous components of the supernatant or filtrate may be accomplished by means of a salt (e.g., ammonium sulfate) or low pH (typically less than 3), followed by purification by a variety of chromatographic procedures (e.g., ion exchange chromatography, affinity chromatography, hydrophobic interaction chromatography, hydrophobic charge induction chromatography etc.) or similar art recognized procedures. It is not intended that the present invention be limited to any particular separation method, as it is contemplated that any method will find use in the present invention.

In certain preferred embodiments, when the expressed desired polypeptide is secreted from the bacterial cells, the polypeptide is purified from the growth media. In preferred embodiments, the expression host cells are removed from the media before purification of the polypeptide (e.g. by centrifugation).

When the expressed recombinant desired polypeptide is not secreted from the host cell, the host cell is preferably disrupted and the polypeptide released into an aqueous “extract” which is the first stage of purification. Preferably, the expression host cells are collected from the media before the cell disruption (e.g. by centrifugation). The cell disruption may be performed by using any suitable means known in the art, such as by lysozyme or beta-glucanase digestion or by forcing the cells through high pressure (See e.g., Scobes, Protein Purification, Second edition, Springer-Verlag)

In some embodiments, other recombinant constructions include the addition of purification facilitating domains to the nucleotide sequence encoding a modified variant BBPI polypeptide domain which facilitates purification of the soluble proteins (Kroll D J et al (1993) DNA Cell Biol 12:441-53). In some embodiments, the addition of six histidine residues (i.e., a “His Tag”) to the C-terminus of the modified variant BBPI is used as an aid in the purification of the desired protein and its fusion analog. Use of the His tags as a purification aid is well known in the art (See e.g., Hengen, TIBS 20:285-286 [1995]). The 6× his-tagged proteins are easily purified using Immobilized Metal ion Affinity Chromatography (IMAC), as known to those skilled in the art. Other purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals (Porath J (1992) Protein Expr Purif 3:263-281), protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Immunex Corp, Seattle Wash.). The inclusion of a cleavable linker sequence such as Factor XA or enterokinase (Invitrogen, San Diego Calif.) between the purification domain and the heterologous protein also find use to facilitate purification.

Thus, any method suitable for recovering the fusion BBPIs of the present invention finds use in the present invention. Indeed, it is not intended that the present invention be limited to any particular purification method.

For some applications, it is of great importance that the protease inhibitors produced using the present invention be highly pure (e.g., having a purity of more than 99%). This is particularly true whenever the desired protein is to be used as a therapeutic, but is also necessary for other applications. The methods described herein provide a way of producing substantially pure desired proteins. The desired proteins described herein are useful in pharmaceutical and personal care compositions. However, it is contemplated that proteins of varying purity levels will be produced using the methods of the present invention and it is not intended that the proteins produced using the present invention be limited to any particular level of purity.

5.8.1 Activation of BBPI During Purification: Protease Inhibitory Activity

In some embodiments of the present invention, after growth during the purification process, the activity of the protein i.e. the protease inhibitory activity, is increased by the addition of chemicals that reduce/oxidize disulfide bonds and/or alter the general redox potential, and/or chemicals that alter solvent properties thus affecting protein conformation and aggregation. In some particularly preferred embodiments, addition of a reagent that reduces disulfide bonds, such as 2-mercaptoethanol, is used to increase activity of the protein. However, as those skilled in the art appreciate, depending purity and buffer composition, other disulfide reducing or oxidizing agents (e.g., DTT, TCEP, reduced and oxidized glutathione, cysteine, cystine, cysteamine, thioglycolate, S₂O₃ ²⁻, S₂O₄ ²⁻, S₂O₅ ²⁻, SO₃ ²⁻, S₂O₇ ²⁻, Cu+, protein disulfide isomerases, protein thiol-disulfide oxidoreductases, etc.), either used alone or in combination, find use in the present invention. Other adjuvants, which alter solvent properties, (e.g. ethanolamine, DMSO, TWEEN®-80, arginine, urea, etc.), are either added alone or preferably in combination with disulfide reducing/oxidizing agents, such as βME, during the purification process also find use in the present invention by increasing the activity of the protein. In certain preferred embodiments, partially purified protein is diluted in buffer (in some particularly preferred embodiments, a zwitterionic buffer with TWEEN®-80 at basic pH) and activated with βME and a disulfide oxidizing agent (in alternative preferred embodiments, oxidized glutathione or sodium sulfite).

In addition, it is contemplated that conditions will be screened in order to determine the optimal activation of the BBPI protein, if desired. For example, various βME concentrations (0.1-10 mM), oxidizing agent concentrations (0 to 1/20 to 20 times the βME concentration) pH (7.5-9.5), temperatures (15-40° C.), dilutions (1-20 fold), incubation times (12-72 h), aeration (incubations under inert gas to vigorous mixing under oxygen containing gases), buffer types (Tris, CHES, CAPS, Tricine, TAPS, other zwitterionic buffers, etc.), buffer concentrations (0.1-1 M), and the addition of various adjuvants known to alter solvent properties thereby affecting protein conformation and aggregation (e.g., ethanolamine, DMSO, TWEEN®-80, arginine, urea, etc.) are tested in order to determine the optimum conditions for the expression system used. It is not intended that the present invention be limited to any particular disulfide reducing/oxidizing agent, dilution, temperature, pH, buffer type or composition, or adjuvant, as any suitable reagents known to those skilled in the art find use in the present invention.

Optimal activation of BBPIs by thiol reducing agents and/or oxidizing agents is monitored by measuring an increase in the protease inhibitory activity of the unmodified variant BBPI. Trypsin inhibitory activity is the protease inhibitory that is assayed in variant BBPIs in which the chymotrypsin loop has been replaced by a variant peptide, while chymotrypsin inhibitory activity is the protease inhibitory that is assayed in variant BBPIs in which the trypsin loop has been replaced by a variant peptide. In some embodiments, the effect of at least one amino acid substitution on the trypsin inhibitory activity of a modified BBPI is assayed and compared to the trypsin inhibitory activity of the unmodified precursor BBPI.

In some embodiments, modified variant BBPIs comprising at least one amino acid substitution have greater trypsin inhibitory activity than that of the unmodified precursor BBPI. In some embodiments, the single amino acid substitution that generates a modified variant BBPI having a greater TIA than the unmodified precursor is chosen from equivalent to a position chosen from positions equivalent to positions 1, 4, 5, 11, 13, 18, 25, 27, 29, 31, 38, 40, 50, 52, 55, and 65 of SEQ ID NO:187 result in the following substituted amino acids. In one embodiment, the substituted amino acid at the amino acid position equivalent to position 1 of SEQ ID NO:187 is chosen from A and C. In another embodiment, the substituted amino acid at the amino acid position equivalent to position 4 of SEQ ID NO:187 is V. In another embodiment, the substituted amino acid at the amino acid position equivalent to position 5 of SEQ ID NO:187 is chosen from P, and A. In another embodiment, the substituted amino acid at the amino acid position equivalent to position 11 of SEQ ID NO:187 is G. In another embodiment, the substituted amino acid at the amino acid position equivalent to position 13 of SEQ ID NO:187 is chosen from Y, I, F, M, L, V, K, and R. In another embodiment, the substituted amino acid at the amino acid position equivalent to position 18 of SEQ ID NO:187 include 1, V and L. In another embodiment, the substituted amino acid at the amino acid position equivalent to position 25 of SEQ ID NO:187 is chosen from K, N, W, I, A and R. In another embodiment, the substituted amino acid at the amino acid position equivalent to position 27 of SEQ ID NO:187 include R, K, V, A, and Q. In another embodiment, the substituted amino acid at the amino acid position equivalent to position 29 of SEQ ID NO:187 is chosen from R, K, and P. In another embodiment, the substituted amino acid at the amino acid position equivalent to position 31 of SEQ ID NO:187 is chosen from Q, H, E, A, R, W, K and T. In another embodiment, the substituted amino acid at the amino acid position equivalent to position 38 of SEQ ID NO:187 is chosen from N, K and R. In another embodiment, the substituted amino acid at the amino acid position equivalent to position 40 of SEQ ID NO:187 is chosen from H, K, Q, R, and Y. In another embodiment, the substituted amino acid at the amino acid position equivalent to position 50 of SEQ ID NO:187 is chosen from R, Q, K, T, V, M, and S. In another embodiment, the substituted amino acid at the amino acid position equivalent to position 52 of SEQ ID NO:187 is chosen from K, T, R, Q, L, H, A, M, S and E. In another embodiment, the substituted amino acid at the amino acid position equivalent to position 55 of SEQ ID NO:187 is M. In another embodiment, the substituted amino acid at the amino acid position equivalent to position 65 of SEQ ID NO:187 is chosen from E, Q, and D.

In some embodiments, the modified variant BBPI that have greater TIA and PY than the corresponding precursor unmodified BBPIs each comprise a combination of two amino acid substitutions at amino acids at positions equivalent to positions 50 and 52 of SEQ ID NO:187. In some embodiments, the combination of two amino acid substitutions is 50T-52A. The invention provides for any one of the variant BBPI scaffolds described in Section 5.3 and further comprising the combination of the two amino acid substitutions 50T-52A, as described in section 5.4. In one embodiment, the chymotrypsin loop of the variant scaffold is a VEGF variant peptide e.g. SEQ ID NO:9, and the variant scaffold is altered further to comprise a combination of three amino acid substitutions at positions equivalent to positions 13, 50 and 52 of SEQ ID NO:187 to generate a modified variant BBPI scaffold. In one embodiment, the modified variant BBPI comprising a combination of two amino acid substitutions is the modified variant BBIt-AV-F50T-V52A of SEQ ID NO:595.

In some embodiments, the modified variant BBPI that have greater TIA and PY than the corresponding precursor unmodified BBPIs each comprise a combination of three amino acid substitutions at amino acids at positions equivalent to positions 13, 50 and 52 of SEQ ID NO:187. In some embodiments, the combination of three amino acid substitutions is chosen from a combination of substitutions at positions 25-50-52, 29-50-52, 40-50-52, and 13-50-52. In some embodiments, the combination of three amino acid substitutions is chosen from 25L-50T-52A, 29P-50T-52A, 40K-50T-52A and 13I-50T-52A. The invention provides for any one of the variant BBPI scaffolds described in 5.3 and further comprising the combination of the three amino acid substitutions chosen from 25L-50T-52A, 29P-50T-52A, 40K-50T-52A and 13I-50T-52A, as described in section 5.4. In one embodiment, the chymotrypsin loop of the variant scaffold is a VEGF variant peptide e.g. SEQ ID NO:9, and the variant scaffold is altered further to comprise a combination of three amino acid substitutions at positions equivalent to positions 13, 50 and 52 of SEQ ID NO:187 to generate a modified variant BBPI scaffold. In one embodiment, the modified variant BBPI comprising a combination of three amino acid substitutions is chosen from the modified variant BBIt-AV-S25L-F50T-V52A of SEQ ID NO: 603, the modified variant BBIt-AV-L29P-F50T-V52A of SEQ ID NO:607, and the modified variant BBIt-AV-A40K-F50T-V52A of SEQ ID NO:609.

In some embodiments, the modified variant BBPI that have greater TIA than the corresponding precursor unmodified BBPIs each comprise a combination of four amino acid substitutions at amino acids at positions equivalent to positions 13, 29, 50 and 52 of SEQ ID NO:187. In some embodiments, the combination of four amino acid substitutions is chosen from a combination of substitutions at positions 13-25-50-52, 13-29-50-52, 25-29-50-52, 13-40-50-52, 25-40-50-52, and 29-40-50-52. In some embodiments, the combination of four amino acid substitutions is chosen from 13I-25L-50T-52A, 13I-29P-50T-52A, 25L-29P-50T-52A, 13I-40K-50T-52A, 25L-40K-50T-52A, and 29P-40K-50T-52A. The invention provides for any one of the variant BBPI scaffolds described in Section 5.3 and further comprising the combination of the four amino acid substitutions chosen from 13I-25L-50T-52A, 13I-29P-50T-52A, 25L-29P-50T-52A, 13I-40K-50T-52A, 25L-40K-50T-52A, and 29P-40K-50T-52A, as described in section 5.4. In one embodiment, the chymotrypsin loop of the variant scaffold is a VEGF variant peptide chosen from SEQ ID NO:9 and 460, and the variant scaffold is altered further to comprise a combination of four amino acid substitutions at positions equivalent to positions 13, 29, 50 and 52 of SEQ ID NO:187 to generate a modified variant BBPI scaffold. In one embodiment, the modified variant BBPI comprising a combination of four amino acid substitutions is chosen from the modified variant BBIt-AV-A13I-S25L-F50T-V52A of SEQ ID NO:596, the modified variant BBIt-AV-A13I-L29P-F50T-V52A of SEQ ID NO:600, the modified variant BBIt-AV-A13I-A40K-F50T-V52A of SEQ ID NO:602, the modified variant BBIt-AV-S25L-L29P-F50T-V52A of SEQ ID NO:604, the modified variant BBIt-AV-S25L-A40K-F50T-V52A of SEQ ID NO:606, the modified variant BBIt-AV-L29P-A40K-F50T-V52A of SEQ ID NO:608, and the modified variant BBIt-VEGKD-A13I-S25K-L29P-V52K of SEQ ID NO:643. In another embodiment, the chymotrypsin loop of the variant scaffold is an FGF5 variant peptide chosen from SEQ ID NOS:433 and 434, and the variant scaffold is altered further to comprise a combination of four amino acid substitutions at positions equivalent to positions 13, 29, 50 and 52 of SEQ ID NO:187 to generate a modified variant BBPI scaffold. In one embodiment, the modified variant BBPI comprising a combination of four amino acid substitutions is chosen from the modified variant BBIt-MM007-Q-A13I-L29P-F50T-V52A of SEQ ID NO:432, and the modified variant BBIt-FGFps2-Q-A13I-L29P-F50T-V52A of SEQ ID NO:434. In another embodiment, the chymotrypsin loop of the variant scaffold is a TGFβ variant peptide chosen from SEQ ID NOS:436, 437 and 438, and the variant scaffold is altered further to comprise a combination of four amino acid substitutions at positions equivalent to positions 13, 29, 50 and 52 of SEQ ID NO:187 to generate a modified variant BBPI scaffold. In one embodiment, the modified variant BBPI comprising a combination of four amino acid substitutions is chosen from the modified variant BBIt-PEN3-Q-A13I-L29P-F50T-V52A of SEQ ID NO:443, the modified variant BBIt-MM021W-Q-A13I-L29P-F50T-V52A of SEQ ID NO:445, and the modified variant BBIt-WTQ-Q-A13I-L29P-F50T-V52A of SEQ ID NO:447.

In some embodiments, the modified variant BBPI that have greater TIA than the corresponding precursor unmodified BBPIs each comprise a combination of five amino acid substitutions at amino acids at positions equivalent to positions 13, 29, 40, 50 and 52 of SEQ ID NO:187. In some embodiments, the combination of five amino acid substitutions is chosen from a combination of substitutions at positions 13-25-29-50-52, 13-29-40-50-52, 13-25-40-50-52, 25-29-40-50-52, 13-29-40-50-52, 13-29-40-50-52, 13-29-40-50-52 and 13-29-40-50-52. In some embodiments, the combination of five amino acid substitutions is chosen from 13I-25L-29P-50T-52A, 13I-29P-40K-50T-52A, 13I-25L-40K-50T-52A, 25L-29P-40K-50T-52A, 13L-29P-40K-50T-52A, 13I-29K-40K-50T-52A, 13I-29P-40K-50K-52A and 13I-29P-40K-50T-52T. The invention provides for any one of the variant BBPI scaffolds described in Section 5.3 and further comprising the combination of the five amino acid substitutions chosen from 13I-25L-29P-50T-52A, 13I-29P-40K-50T-52A, 13I-25L-40K-50T-52A, 25L-29P-40K-50T-52A, 13L-29P-40K-50T-52A, 13I-29K-40K-50T-52A, 13I-29P-40K-50K-52A and 13I-29P-40K-50T-52T, as described in section 5.4. In one embodiment, the chymotrypsin loop of the variant scaffold is a VEGF variant peptide of SEQ ID NO:9, and the variant scaffold is altered further to comprise a combination of five amino acid substitutions at positions equivalent to positions 13, 29, 40, 50 and 52 of SEQ ID NO:187 to generate a modified variant BBPI scaffold. In one embodiment, the modified variant BBPI comprising a combination of five amino acid substitutions is chosen from the modified variant BBIt-AV-A13I-S25L-L29P-F50T-V52A of SEQ ID NO:597, the modified variant BBIt-AV-A13I-L29P-A40K-F50T-V52A of SEQ ID NO:599, the modified variant BBIt-AV-A13I-S25L-A40K-F50T-V52A of SEQ ID NO:601, the modified variant BBIt-AV-S25L-L29P-A40K-F50T-V52A of SEQ ID NO:605, the modified variant BBIt-AV-A13L-L29P-A40K-F50T-V52A of SEQ ID NO:615, the modified variant BBIt-AV-A13I-L29K-A40K-F50T-V52A of SEQ ID NO:620, the modified variant BBIt-AV-A13I-L29P-A40K-F50K-V52A of SEQ ID NO:624, and the modified variant BBIt-AV-A13I-L29P-A40K-F50T-V52T of SEQ ID NO:625.

In some embodiments, the modified variant BBPI that have greater TIA than the corresponding precursor unmodified BBPIs each comprise a combination of six amino acid substitutions at amino acids at positions equivalent to positions 13, 25, 29, 40, 50 and 52 of SEQ ID NO:187. In some embodiments, the combination of six amino acid substitutions is chosen from a combination of substitutions at positions 13-25-29-40-50-52, 1-13-29-40-50-52, 4-13-29-40-50-52, 5-13-29-40-50-52, 11-13-29-40-50-52, 13-25-29-40-50-52, 13-27-29-40-50-52, 13-29-31-40-50-52, 13-29-31-40-50-52, 13-29-38-40-50-52, and 13-29-38-40-50-52. In some embodiments, the combination of six amino acid substitutions is chosen from 13I-25L-29P-40K-50T-52A, 1C-13I-29P-40K-50T-52A, 4V-13I-29P-40K-50T-52A, 5P-13I-29P-40K-50T-52A, 11G-13I-29P-40K-50T-52A, 13I-25R-29P-40K-50T-52A, 13I-27R-29P-40K-50T-52A, 13I-29P-31A-40K-50T-52A, 13I-29P-31R-40K-50T-52A, 13I-29P-38N-40K-50T-52A, and 13I-29P-38N-40K-50T-52A. The invention provides for any one of the variant BBPI scaffolds described in Section 5.3 and further comprising the combination of the six amino acid substitutions chosen from 13I-25L-29P-40K-50T-52A, 1C-13I-29P-40K-50T-52A, 4V-13I-29P-40K-50T-52A, 5P-13I-29P-40K-50T-52A, 11G-13I-29P-40K-50T-52A, 13I-25R-29P-40K-50T-52A, 13I-27R-29P-40K-50T-52A, 13I-29P-31A-40K-50T-52A, 13I-29P-31R-40K-50T-52A, 13I-29P-38N-40K-50T-52A, and 13I-29P-38N-40K-50T-52A, as described in section 5.4. In one embodiment, the chymotrypsin loop of the variant scaffold is a VEGF variant peptide of SEQ ID NO:9, and the variant scaffold is altered further to comprise a combination of six amino acid substitutions at positions equivalent to positions 13, 25, 29, 40, 50 and 52 of SEQ ID NO:187 to generate a modified variant BBPI scaffold. In one embodiment, the modified variant BBPI comprising a combination of six amino acid substitutions is chosen from the modified variant BBIt-AV-A13I-S25L-L29P-A40K-F50T-V52A of SEQ ID NO:598, the modified variant BBIt-AV-D1C-A13I-L29P-A40K-F50T-V52A of SEQ ID NO:611, the modified variant BBIt-AV-S4V-A13I-L29P-A40K-F50T-V52A of SEQ ID NO:612, the modified variant BBIt-AV-S5P-A13I-L29P-A40K-F50T-V52A of SEQ ID NO:613, the modified variant BBIt-AV-Q11G-A13I-L29P-A40K-F50T-V52A of SEQ ID NO:614, the modified variant BBIt-AV-A13I-S25R-L29P-A40K-F50T-V52A- of SEQ ID NO:616, the modified variant BBIt-AV-A13I-M27R-L29P-A40K-F50T-V52A of SEQ ID NO:619, the modified variant BBIt-AV-A13I-L29P-S31A-A40K-F50T-V52A of SEQ ID NO:621, the modified variant BBIt-AV-A13I-L29P-S31R-A40K-F50T-V52A of SEQ ID NO:622, the modified variant BBIt-AV-A13I-L29P-S38N-A40K-F50T-V52A of SEQ ID NO:623, and the modified variant BBIt-AV-A13I-L29P-S38N-A40K-F50T-V52A of SEQ ID NO:626.

In some embodiments, the modified variant BBPI that have greater TIA than the corresponding precursor unmodified BBPIs each comprise a combination of seven amino acid substitutions at amino acids at positions equivalent to positions 13, 25, 29, 31, 40, 50 and 52 of SEQ ID NO:187. In some embodiments, the combination of seven amino acid substitutions is chosen from a combination of substitutions at positions 13-25-29-31-40-50-52, 13-25-29-31-40-50-52, 13-25-27-29-31-50-52, 13-25-27-29-31-50-52, 13-25-27-29-31-50-52, 13-25-27-29-31-50-52, 13-25-27-29-31-50-52, 13-25-27-29-31-50-52, 13-25-27-29-31-50-52, 13-25-27-29-31-50-52, and 13-25-27-29-31-50-52. In some embodiments, the combination of seven amino acid substitutions is chosen from 13L-25R-29P-31A-40K-50T-52A, 13L-25R-29P-31R-40K-50T-52A, 13I-25R-27A-29P-31A-50K-52T, 13I-25R-27A-29P-31A-50K-52T, 13I-25R-27A-29P-31A-50K-52T, 13I-25R-27A-29P-31A-50K-52T, 13I-25R-27A-29P-31A-50K-52T, 13I-25R-27A-29P-31A-50K-52T, 13I-25R-27A-29P-31A-50K-52T, 13I-25R-27A-29P-31A-50K-52T, and 13I-25R-27A-29P-31A-50K-52T. The invention provides for any one of the variant BBPI scaffolds described in Section 5.3 and further comprising the combination of the six amino acid substitutions chosen from 13L-25R-29P-31A-40K-50T-52A, 13L-25R-29P-31R-40K-50T-52A, 13I-25R-27A-29P-31A-50K-52T, 13I-25R-27A-29P-31A-50K-52T, 13I-25R-27A-29P-31A-50K-52T, 13I-25R-27A-29P-31A-50K-52T, 13I-25R-27A-29P-31A-50K-52T, 13I-25R-27A-29P-31A-50K-52T, 13I-25R-27A-29P-31A-50K-52T, 13I-25R-27A-29P-31A-50K-52T, and 13I-25R-27A-29P-31A-50K-52T, as described in section 5.4. In one embodiment, the chymotrypsin loop of the variant scaffold is a VEGF variant peptide of SEQ ID NO:9, and the variant scaffold is altered further to comprise a combination of seven amino acid substitutions at positions equivalent to positions 13, 25, 29, 31, 40, 50 and 52 of SEQ ID NO:187 to generate a modified variant BBPI scaffold. In one embodiment, the modified variant BBPI comprising a combination of seven amino acid substitutions is chosen from the modified variant BBIt-AV-A13I-S25R-L29P-S31A-A40K-F50T-V52A of SEQ ID NO:617, the modified variant BBIt-AV-A13L-S25R-L29P-S31R-A40K-F50T-V52A of SEQ ID NO:618, the modified variant of BBIt-VEGF-V1-A13I-S25R-M27A-L29P-S31A-F50K-V52T SEQ ID NO:491, the modified variant BBIt-VEGF-V2-A13I-S25R-M27A-L29P-S31A-F50K-V52T of SEQ ID NO:632, the modified variant BBIt-VEGF-V3-A13I-S25R-M27A-L29P-S31A-F50K-V52T of SEQ ID NO:633, the modified variant BBIt-VEGF-V4-A13I-S25R-M27A-L29P-S31A-F50K-V52T of SEQ ID NO:634, the modified variant BBIt-VEGF-V5-A13I-S25R-M27A-L29P-S31A-F50K-V52T of SEQ ID NO:635, the modified variant BBIt-VEGF-V6-A13I-S25R-M27A-L29P-S31A-F50K-V52T of SEQ ID NO:636, the modified variant BBIt-TNFα-T1-A13I-S25R-M27A-L29P-S31A-F50K-V52T of SEQ ID NO:637, the modified variant BBIt-TNFα-T2-A13I-S25R-M27A-L29P-S31A-F50K-V52T of SEQ ID NO:638, and the modified variant BBIt-TNFα-T3-A13I-S25R-M27A-L29P-S31A-F50K-V52T of SEQ ID NO:639.

In some embodiments, the modified variant BBPI that have greater TIA than the corresponding precursor unmodified BBPIs each comprise a combination of eight amino acid substitutions at amino acids at positions equivalent to positions 13, 25, 27, 29, 31, 40, 50 and 52 of SEQ ID NO:187. In some embodiments, the combination of eight amino acid substitutions is chosen from a combination of substitutions at positions 13-25-27-29-31-40-50-52, 13-25-27-29-31-40-50-52, 13-25-27-29-31-40-50-52, 13-25-27-29-31-40-50-52, and 13-25-27-29-31-40-50-52. In some embodiments, the combination of eight amino acid substitutions is chosen from combinations 13I-25R-27A-29P-31A-40H-50K-52T, 13I-25K-27A-29R-31E-40K-50Q-52Q, 13I-25K-27R-29E-31A-40H-50R-52K, 13I-25K-27A-29R-31A-40H-50R-52L, and 13I-25K-27Q-29P-31E-40H-50R-52Q. The invention provides for any one of the variant BBPI scaffolds described in Section 5.3 and further comprising the combination of the eight amino acid substitutions chosen from combinations 13I-25R-27A-29P-31A-40H-50K-52T, 13I-25K-27A-29R-31E-40K-50Q-52Q, 13I-25K-27R-29E-31A-40H-50R-52K, 13I-25K-27A-29R-31A-40H-50R-52L, and 13I-25K-27Q-29P-31E-40H-50R-52Q, as described in section 5.4. In one embodiment, the chymotrypsin loop of the variant scaffold is a VEGF variant peptide of SEQ ID NO:9, and the variant scaffold is altered further to comprise a combination of seven amino acid substitutions at positions equivalent to positions 13, 25, 27, 29, 31, 40, 50 and 52 of SEQ ID NO:187 to generate a modified variant BBPI scaffold. In one embodiment, the modified variant BBPI comprising a combination of eight amino acid substitutions is chosen from the modified variant BBIt-AV-A13I-S25R-M27A-L29P-S31A-A40H-F50K-V52T of SEQ ID NO:627, the modified variant of BBIt-AV-A13I-S25K-M27A-L29R-S31E-A40K-F50Q-V52Q of SEQ ID NO:628, the modified variant BBIt-AV-A13I-S25K-M27R-L29E-S31A-A40H-F50R-V52K of SEQ ID NO:629, the modified variant BBIt-AV-A13I-S25K-M27A-L29R-S31A-A40H-F50R-V52L of SEQ ID NO:630, and the modified variant BBIt-AV-A13I-S25K-M27Q-L29P-S31E-A40H-F50R-V52Q of SEQ ID NO:631.

In some embodiments, the modified variant BBPI that have greater TIA than the corresponding precursor unmodified BBPIs each further comprise a peptide insert that is positioned at the N-terminus of the modified variant BBPI. In some embodiments, the peptide insert comprises a sequence of between 1 and 15 amino acids. In other embodiments, the peptide insert comprises a sequence between 5 and 10 amino acids. In some embodiments, the peptide insert comprises the peptide of SEQ ID NO:389 (DDEPSKPCCDPDP; SEQ ID NO:389). Examples of modified variant BBPIs that the peptide insert of SEQ ID NO:389 are the modified variant 4D13BBIt-AV of SEQ ID NO:390 (DDEPSKPCCDPDPDDESSKPCCDQCACTKSNPPQCRCSDMRLNSCHSACKSCACYNLYGWTCFCVDITDFCYEPCKPSE; SEQ ID NO:390), and the modified variant BBIt-AV-4D13-13I-29P-40K-50T-52A of SEQ ID NO: 413.

One measure of enhancement in trypsin and/or chymotrypsin inhibitory activity can be determined as the increase in an enzymatic activity ratio i.e. BCE:BBPI, in the modified variant BBPI when compared to that of the unmodified variant BBPI. The BCE:BBPI enzymatic activity ratio is the ratio of BBPI protease inhibitory activity e.g. trypsin inhibitory activity, to the BCE i.e. cellulase enzymatic activity. The ratio of the unmodified variant BBPI is assigned a value of 1. A ratio equal or greater than 1 for a modified variant BBPI indicates that the modified variant BBPI has a greater protease inhibitory activity e.g. trypsin inhibitory activity than that of the unmodified precursor BBPI. In some embodiments, the activity ratio of the modified variant BBPI is at least 1, at least about 1.05, about at least about 1.1, at least about 1.2, at least about 1.3, at least about 1.4, at least about 1.5, at least about 1.6, at least about 1.7, at least about 1.8. at least about 1.9, and at least about 2. In other embodiments, the activity ratio is at least about 2.1, at least about 2.2, at least about 2.3, at least about 2.4, at least about 2.5, at least about 2.6, at least about 2.7, at least about 2.8, at least about 2.9 and at least about 3. In yet other embodiments, the activity ratio is at least about 3.5, at least about 4.0, and at least about 5. Thus, in some embodiments, the protease inhibitory activity e.g. protease inhibitory activity, of the modified variant BBPI greater than that of the corresponding unmodified precursor BBPI by at least about 0.5%, about 1.0%, about 1.5%, about 2.0%, about 2.5%, about 3.0%, about 4.0%, about 5.0%, about 8.0%, about 10%, about 15%, about 20%, about 25%, about 30%, about 40%, about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100% or more. In other embodiments, the protease inhibitory activity e.g. protease inhibitory activity, of the modified variant BBPI greater than that of the corresponding unmodified precursor BBPI by at least about 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, and up to at least about 200%.

In other embodiments, modified variant BBPIs comprising a combination of amino acid substitutions have greater trypsin inhibitory activity than that of the unmodified precursor BBPI. In some embodiments, the at least two, at least three, at least four, at least five, at least six, at least seven, and at least eight amino acid substitutions generate modified variant BBPIs that have greater TIA than the unmodified precursor variant BBPI. All modified variant BBPIs comprising a combination of amino acid substitutions as described in section 5.4 have greater trypsin inhibitory activity than the corresponding unmodified precursor BBPIs.

5.8.2 BBPI Production Yield

In some embodiments, the amino acid substitutions made in the precursor variant BBPI are assessed for the ability of the resulting modified variant BBPI to have a greater production yield that is greater than that at which the unmodified precursor BBPI is produced i.e. the modified variant BBPI is produced at a level that is greater than that at which the unmodified precursor BBPI is produced. In some embodiments, the invention provides for modified variant BBPIs comprising a combination of amino acid substitutions as described in section 5.4 that have greater protease inhibitory activity e.g. trypsin inhibitory activity, and greater production yield than the corresponding unmodified precursor BBPIs.

Modified variant BBPIs that have greater TIA and PY than the corresponding precursor unmodified BBPIs each comprise at least two, at least three, at least four, at least five, at least six, at least seven, and at least eight amino acid substitutions (see Examples 12, 13, 14 and 15).

In some embodiments, the modified variant BBPI that have greater TIA and PY than the corresponding precursor unmodified BBPIs each comprise a combination of two amino acid substitutions at amino acids at positions equivalent to positions 50 and 52 of SEQ ID NO:187. In some embodiments, the combination of two amino acid substitutions is 50T-52A. The invention provides for any one of the variant BBPI scaffolds described in Section 5.3 and further comprising the combination of the two amino acid substitutions 50T-52A, as described in section 5.4. In one embodiment, the chymotrypsin loop of the variant scaffold is a VEGF variant peptide e.g. SEQ ID NO:9, and the variant scaffold is altered further to comprise a combination of three amino acid substitutions at positions equivalent to positions 13, 50 and 52 of SEQ ID NO:187 to generate a modified variant BBPI scaffold. In one embodiment, the modified variant BBPI comprising a combination of two amino acid substitutions is the modified variant BBIt-AV-F50T-V52A of SEQ ID NO: 6595.

In some embodiments, the modified variant BBPI that have greater TIA and PY than the corresponding precursor unmodified BBPIs each comprise a combination of three amino acid substitutions at amino acids at positions equivalent to positions 13, 50 and 52 of SEQ ID NO:187. In some embodiments, the combination of three amino acid substitutions is chosen from a combination of substitutions at positions 25-50-52, 29-50-52, 40-50-52, and 13-50-52. In some embodiments, the combination of three amino acid substitutions is chosen from 25L-50T-52A, 29P-50T-52A, 40K-50T-52A and 13I-50T-52A. The invention provides for any one of the variant BBPI scaffolds described in Section 5.3 and further comprising the combination of the three amino acid substitutions chosen from 25L-50T-52A, 29P-50T-52A, 40K-50T-52A and 13I-50T-52A, as described in section 5.4. In one embodiment, the chymotrypsin loop of the variant scaffold is a VEGF variant peptide e.g. SEQ ID NO:9, and the variant scaffold is altered further to comprise a combination of three amino acid substitutions at positions equivalent to positions 13, 50 and 52 of SEQ ID NO:187 to generate a modified variant BBPI scaffold. In one embodiment, the modified variant BBPI comprising a combination of three amino acid substitutions is chosen from the modified variant BBIt-AV-S25L-F50T-V52A of SEQ ID NO: 603, the modified variant BBIt-AV-L29P-F50T-V52A of SEQ ID NO:607, and the modified variant BBIt-AV-A40K-F50T-V52A of SEQ ID NO:609.

In some embodiments, the modified variant BBPI that have greater TIA and PY than the corresponding precursor unmodified BBPIs each comprise a combination of four amino acid substitutions at amino acids at positions equivalent to positions 13, 29, 50 and 52 of SEQ ID NO:187. In some embodiments, the combination of four amino acid substitutions is chosen from a combination of substitutions at positions 13-25-50-52, 13-29-50-52, 25-29-50-52, 13-40-50-52, 25-40-50-52, and 29-40-50-52. In some embodiments, the combination of four amino acid substitutions is chosen from 13I-25L-50T-52A, 13I-29P-50T-52A, 25L-29P-50T-52A, 13I-40K-50T-52A, 25L-40K-50T-52A, and 29P-40K-50T-52A. The invention provides for any one of the variant BBPI scaffolds described in Section 5.3 and further comprising the combination of the four amino acid substitutions chosen from 13I-25L-50T-52A, 13I-29P-50T-52A, 25L-29P-50T-52A, 13I-40K-50T-52A, 25L-40K-50T-52A, and 29P-40K-50T-52A, as described in section 5.4. In one embodiment, the chymotrypsin loop of the variant scaffold is a VEGF variant peptide chosen from SEQ ID NO:9 and 460, and the variant scaffold is altered further to comprise a combination of four amino acid substitutions at positions equivalent to positions 13, 29, 50 and 52 of SEQ ID NO:187 to generate a modified variant BBPI scaffold. In one embodiment, the modified variant BBPI comprising a combination of four amino acid substitutions is chosen from the modified variant BBIt-AV-A13I-S25L-F50T-V52A of SEQ ID NO:596, the modified variant BBIt-AV-A13I-L29P-F50T-V52A of SEQ ID NO:600, the modified variant BBIt-AV-A13I-A40K-F50T-V52A of SEQ ID NO:602, the modified variant BBIt-AV-S25L-L29P-F50T-V52A of SEQ ID NO:604, the modified variant BBIt-AV-S25L-A40K-F50T-V52A of SEQ ID NO:606, the modified variant BBIt-AV-L29P-A40K-F50T-V52A of SEQ ID NO:608, and the modified variant BBIt-VEGKD-A13I-S25K-L29P-V52K of SEQ ID NO:643. In another embodiment, the chymotrypsin loop of the variant scaffold is an FGF5 variant peptide chosen from SEQ ID NOS:430 and 431, and the variant scaffold is altered further to comprise a combination of four amino acid substitutions at positions equivalent to positions 13, 29, 50 and 52 of SEQ ID NO:187 to generate a modified variant BBPI scaffold. In one embodiment, the modified variant BBPI comprising a combination of four amino acid substitutions is chosen from the modified variant BBIt-MM007-Q-A13I-L29P-F50T-V52A of SEQ ID NO:432, and the modified variant BBIt-FGFps2-Q-A13I-L29P-F50T-V52A of SEQ ID NO:434. In another embodiment, the chymotrypsin loop of the variant scaffold is a TGFβ variant peptide chosen from SEQ ID NOS:436, 437 and 438, and the variant scaffold is altered further to comprise a combination of four amino acid substitutions at positions equivalent to positions 13, 29, 50 and 52 of SEQ ID NO:187 to generate a modified variant BBPI scaffold. In one embodiment, the modified variant BBPI comprising a combination of four amino acid substitutions is chosen from the modified variant BBIt-PEN3-Q-A13I-L29P-F50T-V52A of SEQ ID NO:443, the modified variant BBIt-MM021W-Q-A13I-L29P-F50T-V52A of SEQ ID NO:445, and the modified variant BBIt-WTQ-Q-A13I-L29P-F50T-V52A of SEQ ID NO:447.

In some embodiments, the modified variant BBPI that have greater TIA and PY than the corresponding precursor unmodified BBPIs each comprise a combination of five amino acid substitutions at amino acids at positions equivalent to positions 13, 29, 40, 50 and 52 of SEQ ID NO:187. In some embodiments, the combination of five amino acid substitutions is chosen from a combination of substitutions at positions 13-25-29-50-52, 13-29-40-50-52, 13-25-40-50-52, 25-29-40-50-52, 13-29-40-50-52, 13-29-40-50-52, 13-29-40-50-52 and 13-29-40-50-52. In some embodiments, the combination of five amino acid substitutions is chosen from 13I-25L-29P-50T-52A, 13I-29P-40K-50T-52A, 13I-25L-40K-50T-52A, 25L-29P-40K-50T-52A, 13L-29P-40K-50T-52A, 13I-29K-40K-50T-52A, 13I-29P-40K-50K-52A and 13I-29P-40K-50T-52T. The invention provides for any one of the variant BBPI scaffolds described in Section 5.3 and further comprising the combination of the five amino acid substitutions chosen from 13I-25L-29P-50T-52A, 13I-29P-40K-50T-52A, 13I-25L-40K-50T-52A, 25L-29P-40K-50T-52A, 13L-29P-40K-50T-52A, 13I-29K-40K-50T-52A, 13I-29P-40K-50K-52A and 13I-29P-40K-50T-52T, as described in section 5.4. In one embodiment, the chymotrypsin loop of the variant scaffold is a VEGF variant peptide of SEQ ID NO:9, and the variant scaffold is altered further to comprise a combination of five amino acid substitutions at positions equivalent to positions 13, 29, 40, 50 and 52 of SEQ ID NO:187 to generate a modified variant BBPI scaffold. In one embodiment, the modified variant BBPI comprising a combination of five amino acid substitutions is chosen from the modified variant BBIt-AV-A13I-S25L-L29P-F50T-V52A of SEQ ID NO:597, the modified variant BBIt-AV-A13I-L29P-A40K-F50T-V52A of SEQ ID NO:599, the modified variant BBIt-AV-A13I-S25L-A40K-F50T-V52A of SEQ ID NO:601, the modified variant BBIt-AV-S25L-L29P-A40K-F50T-V52A of SEQ ID NO:605, the modified variant BBIt-AV-A13L-L29P-A40K-F50T-V52A of SEQ ID NO:615, the modified variant BBIt-AV-A13I-L29K-A40K-F50T-V52A of SEQ ID NO:620, the modified variant BBIt-AV-A13I-L29P-A40K-F50K-V52A of SEQ ID NO:624, and the modified variant BBIt-AV-A13I-L29P-A40K-F50T-V52T of SEQ ID NO:625.

In some embodiments, the modified variant BBPI that have greater TIA and PY than the corresponding precursor unmodified BBPIs each comprise a combination of six amino acid substitutions at amino acids at positions equivalent to positions 13, 25, 29, 40, 50 and 52 of SEQ ID NO:187. In some embodiments, the combination of six amino acid substitutions is chosen from a combination of substitutions at positions 13-25-29-40-50-52, 1-13-29-40-50-52, 4-13-29-40-50-52, 5-13-29-40-50-52, 11-13-29-40-50-52, 13-25-29-40-50-52, 13-27-29-40-50-52, 13-29-31-40-50-52, 13-29-31-40-50-52, 13-29-38-40-50-52, and 13-29-38-40-50-52. In some embodiments, the combination of six amino acid substitutions is chosen from 13I-25L-29P-40K-50T-52A, 1C-13I-29P-40K-50T-52A, 4V-13I-29P-40K-50T-52A, 5P-13I-29P-40K-50T-52A, 11G-13I-29P-40K-50T-52A, 13I-25R-29P-40K-50T-52A, 13I-27R-29P-40K-50T-52A, 13I-29P-31A-40K-50T-52A, 13I-29P-31R-40K-50T-52A, 13I-29P-38N-40K-50T-52A, and 13I-29P-38N-40K-50T-52A. The invention provides for any one of the variant BBPI scaffolds described in Section 5.3 and further comprising the combination of the six amino acid substitutions chosen from 13I-25L-29P-40K-50T-52A, 1C-13I-29P-40K-50T-52A, 4V-13I-29P-40K-50T-52A, 5P-13I-29P-40K-50T-52A, 11G-13I-29P-40K-50T-52A, 13I-25R-29P-40K-50T-52A, 13I-27R-29P-40K-50T-52A, 13I-29P-31A-40K-50T-52A, 13I-29P-31R-40K-50T-52A, 13I-29P-38N-40K-50T-52A, and 13I-29P-38N-40K-50T-52A, as described in section 5.4. In one embodiment, the chymotrypsin loop of the variant scaffold is a VEGF variant peptide of SEQ ID NO:9, and the variant scaffold is altered further to comprise a combination of six amino acid substitutions at positions equivalent to positions 13, 25, 29, 40, 50 and 52 of SEQ ID NO:187 to generate a modified variant BBPI scaffold. In one embodiment, the modified variant BBPI comprising a combination of six amino acid substitutions is chosen from the modified variant BBIt-AV-A13I-S25L-L29P-A40K-F50T-V52A of SEQ ID NO:598, the modified variant BBIt-AV-D1C-A13I-L29P-A40K-F50T-V52A of SEQ ID NO:611, the modified variant BBIt-AV-S4V-A13I-L29P-A40K-F50T-V52A of SEQ ID NO:612, the modified variant BBIt-AV-S5P-A13I-L29P-A40K-F50T-V52A of SEQ ID NO:613, the modified variant BBIt-AV-Q11G-A13I-L29P-A40K-F50T-V52A of SEQ ID NO:614, the modified variant BBIt-AV-A13I-S25R-L29P-A40K-F50T-V52A- of SEQ ID NO:616, the modified variant BBIt-AV-A13I-M27R-L29P-A40K-F50T-V52A of SEQ ID NO:619, the modified variant BBIt-AV-A13I-L29P-S31A-A40K-F50T-V52A of SEQ ID NO:621, the modified variant BBIt-AV-A13I-L29P-S31R-A40K-F50T-V52A of SEQ ID NO:622, the modified variant BBIt-AV-A13I-L29P-S38N-A40K-F50T-V52A of SEQ ID NO:623, and the modified variant BBIt-AV-A13I-L29P-S38N-A40K-F50T-V52A of SEQ ID NO:626.

In some embodiments, the modified variant BBPI that have greater TIA and PY than the corresponding precursor unmodified BBPIs each comprise a combination of seven amino acid substitutions at amino acids at positions equivalent to positions 13, 25, 29, 31, 40, 50 and 52 of SEQ ID NO:187. In some embodiments, the combination of seven amino acid substitutions is chosen from a combination of substitutions at positions 13-25-29-31-40-50-52, 13-25-29-31-40-50-52, 13-25-27-29-31-50-52, 13-25-27-29-31-50-52, 13-25-27-29-31-50-52, 13-25-27-29-31-50-52, 13-25-27-29-31-50-52, 13-25-27-29-31-50-52, 13-25-27-29-31-50-52, 13-25-27-29-31-50-52, and 13-25-27-29-31-50-52. In some embodiments, the combination of seven amino acid substitutions is chosen from 13L-25R-29P-31A-40K-50T-52A, 13L-25R-29P-31R-40K-50T-52A, 13I-25R-27A-29P-31A-50K-52T, 13I-25R-27A-29P-31A-50K-52T, 13I-25R-27A-29P-31A-50K-52T, 13I-25R-27A-29P-31A-50K-52T, 13I-25R-27A-29P-31A-50K-52T, 13I-25R-27A-29P-31A-50K-52T, 13I-25R-27A-29P-31A-50K-52T, 13I-25R-27A-29P-31A-50K-52T, and 13I-25R-27A-29P-31A-50K-52T. The invention provides for any one of the variant BBPI scaffolds described in Section 5.3 and further comprising the combination of the six amino acid substitutions chosen from 13L-25R-29P-31A-40K-50T-52A, 13L-25R-29P-31R-40K-50T-52A, 13I-25R-27A-29P-31A-50K-52T, 13I-25R-27A-29P-31A-50K-52T, 13I-25R-27A-29P-31A-50K-52T, 13I-25R-27A-29P-31A-50K-52T, 13I-25R-27A-29P-31A-50K-52T, 13I-25R-27A-29P-31A-50K-52T, 13I-25R-27A-29P-31A-50K-52T, 13I-25R-27A-29P-31A-50K-52T, and 13I-25R-27A-29P-31A-50K-52T, as described in section 5.4. In one embodiment, the chymotrypsin loop of the variant scaffold is a VEGF variant peptide of SEQ ID NO:9, and the variant scaffold is altered further to comprise a combination of seven amino acid substitutions at positions equivalent to positions 13, 25, 29, 31, 40, 50 and 52 of SEQ ID NO:187 to generate a modified variant BBPI scaffold. In one embodiment, the modified variant BBPI comprising a combination of seven amino acid substitutions is chosen from the modified variant BBIt-AV-A13I-S25R-L29P-S31A-A40K-F50T-V52A of SEQ ID NO:617, the modified variant BBIt-AV-A13I-S25R-L29P-S31R-A40K-F50T-V52A of SEQ ID NO:618, the modified variant of BBIt-VEGF-V1-A13I-S25R-M27A-L29P-S31A-F50K-V52T SEQ ID NO:491, the modified variant BBIt-VEGF-V2-A13I-S25R-M27A-L29P-S31A-F50K-V52T of SEQ ID NO:632, the modified variant BBIt-VEGF-V3-A13I-S25R-M27A-L29P-S31A-F50K-V52T of SEQ ID NO:633, the modified variant BBIt-VEGF-V4-A13I-S25R-M27A-L29P-S31A-F50K-V52T of SEQ ID NO:634, the modified variant BBIt-VEGF-V5-A13I-S25R-M27A-L29P-S31A-F50K-V52T of SEQ ID NO:635, the modified variant BBIt-VEGF-V6-A13I-S25R-M27A-L29P-S31A-F50K-V52T of SEQ ID NO:636, the modified variant BBIt-TNFα-T1-A13I-S25R-M27A-L29P-S31A-F50K-V52T of SEQ ID NO:637, the modified variant BBIt-TNFα-T2-A13I-S25R-M27A-L29P-S31A-F50K-V52T of SEQ ID NO:638, and the modified variant BBIt-TNFα-T3-A13I-S25R-M27A-L29P-S31A-F50K-V52T of SEQ ID NO:639.

In some embodiments, the modified variant BBPI that have greater TIA and PY than the corresponding precursor unmodified BBPIs each comprise a combination of eight amino acid substitutions at amino acids at positions equivalent to positions 13, 25, 27, 29, 31, 40, 50 and 52 of SEQ ID NO:187. In some embodiments, the combination of eight amino acid substitutions is chosen from a combination of substitutions at positions 13-25-27-29-31-40-50-52, 13-25-27-29-31-40-50-52, 13-25-27-29-31-40-50-52, 13-25-27-29-31-40-50-52, and 13-25-27-29-31-40-50-52. In some embodiments, the combination of eight amino acid substitutions is chosen from combinations 13I-25R-27A-29P-31A-40H-50K-52T, 13I-25K-27A-29R-31E-40K-50Q-52Q, 13I-25K-27R-29E-31A-40H-50R-52K, 13I-25K-27A-29R-31A-40H-50R-52L, and 13I-25K-27Q-29P-31E-40H-50R-52Q. The invention provides for any one of the variant BBPI scaffolds described in Section 5.3 and further comprising the combination of the eight amino acid substitutions chosen from combinations 13I-25R-27A-29P-31A-40H-50K-52T, 13I-25K-27A-29R-31E-40K-50Q-52Q, 13I-25K-27R-29E-31A-40H-50R-52K, 13I-25K-27A-29R-31A-40H-50R-52L, and 13I-25K-27Q-29P-31E-40H-50R-52Q, as described in section 5.4. In one embodiment, the chymotrypsin loop of the variant scaffold is a VEGF variant peptide of SEQ ID NO:9, and the variant scaffold is altered further to comprise a combination of seven amino acid substitutions at positions equivalent to positions 13, 25, 27, 29, 31, 40, 50 and 52 of SEQ ID NO:187 to generate a modified variant BBPI scaffold. In one embodiment, the modified variant BBPI comprising a combination of eight amino acid substitutions is chosen from the modified variant BBIt-AV-A13I-S25R-M27A-L29P-S31A-A40H-F50K-V52T of SEQ ID NO:627, the modified variant of BBIt-AV-A13I-S25K-M27A-L29R-S31E-A40K-F50Q-V52Q of SEQ ID NO:628, the modified variant BBIt-AV-A13I-S25K-M27R-L29E-S31A-A40H-F50R-V52K of SEQ ID NO:629, the modified variant BBIt-AV-A13I-S25K-M27A-L29R-S31A-A40H-F50R-V52L of SEQ ID NO:630, and the modified variant BBIt-AV-A13I-S25K-M27Q-L29P-S31E-A40H-F50R-V52Q of SEQ ID NO:631.

In some embodiments, the modified variant BBPIs further comprise a peptide insert that is positioned at the N-terminus of the modified variant BBPI. In some embodiments, the peptide insert comprises a sequence of between 1 and 15 amino acids. In other embodiments, the peptide insert comprises a sequence between 5 and 10 amino acids. In some embodiments, the peptide insert comprises the peptide of SEQ ID NO:389. Examples of modified variant BBPIs that the peptide insert of SEQ ID NO:389 are the modified variant 4D13BBIt-AV of SEQ ID NO:390, and the modified variant BBIt-AV-4D13-13I-29P-40K-50T-52A of SEQ ID NO: 413.

The invention further provides for modified variant BBPIs that have greater TIA and PY than the corresponding precursor unmodified BBPIs each comprise any one combination of the amino acid substitutions described above and that have greater protease inhibitory activity than the unmodified precursor variant BBPI. In some embodiments, modified variant BBPIs which contain a variant peptide in place of the chymotrypsin loop of the corresponding precursor unmodified BBPI have greater trypsin inhibitory activity (TIA) than that of the precursor unmodified BBPI scaffold. In other embodiments, modified variant BBPIs which contain a variant peptide in place of the trypsin loop of the corresponding precursor unmodified BBPI have greater chymotrypsin inhibitory activity (TIA) than that of the precursor unmodified BBPI scaffold.

As shown in the Examples, substitutions of at least one amino acid in the backbone of the variant BBPI generates a modified variant BBPI that has a greater production yield that the unmodified variant BBPI. In some embodiments, BBPIs that comprise a combination of two, three, four, five, six, seven or eight amino acid substitutions have a greater production yield than the unmodified precursor BBPI. Thus, the invention provides for modified variant BBPIs that comprise any one combination of the amino acid substitutions described above and that have greater production yield (PY) than the unmodified precursor variant BBPIs. In yet other embodiments, the invention provides for modified variant BBPIs that comprise any one combination of the amino acid substitutions described above and that have greater trypsin inhibitory activity and greater production yield than the TIA and PY of the unmodified precursor variant BBPIs.

In some embodiments, the modified variant BBPIs that have greater TIA and PY than the corresponding precursor unmodified BBPIs each comprise further comprise a peptide insert that is positioned at the N-terminus of the modified variant BBPI. In some embodiments, the peptide insert comprises a sequence of between 1 and 15 amino acids. In other embodiments, the peptide insert comprises a sequence between 5 and 10 amino acids. In some embodiments, the peptide insert comprises the peptide of SEQ ID NO:389. Examples of modified variant BBPIs include the modified variant 4D13BBIt-AV of SEQ ID NO:390, and the modified variant BBIt-AV-4D13-13I-29P-40K-50T-52A of SEQ ID NO: 413.

One measure of enhancement in production yield can be determined as the increase in the level of free BBPI following cleavage of the BBPI from the C-terminus of the BCE core. As described above, in some embodiments, the BBPI fusion proteins can be cleaved using proteases or by chemical means. In other embodiments, the BBPI can be cleaved from the BBPI fusion protein by acid/heat treatment, which cleaves the A-P bonds present in the CBD linker that joins the BCE to the BBPI. In yet other embodiments, the BBPI fusion proteins can be cleaved using glutamyl endopeptidase I from B. licheniformis. In some embodiments, the enhancement in production yield of a modified variant BBPI is measured as the increase in the level of free modified variant BBPI following activation of the fusion BBPI and following acid heat treatment of the BBPI fusion protein, when compared to the level of free unmodified precursor BBPI that was subjected to the same treatment. In other embodiments, the enhancement in production yield of a modified variant BBPI is measured as the increase in the level of free modified variant BBPI without activation of the fusion BBPI and following acid heat treatment of the BBPI fusion protein, when compared to the level of the corresponding free unmodified precursor BBPI that was subjected to the same treatment. In some embodiments, the free modified variant BBPI has a greater production yield and a greater TIA than the corresponding precursor BBPI.

In some embodiments, the production yield of the modified variant BBPI is greater than that of the corresponding unmodified precursor BBPI by at least about 0.5%, about 1.0%, about 1.5%, about 2.0%, about 2.5%, about 3.0%, about 4.0%, about 5.0%, about 8.0%, about 10%, about 15%, about 20%, about 25%, about 30%, about 40%, about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100% or more. In other embodiments, the production yield of the modified variant BBPI is greater than that of the corresponding unmodified precursor BBPI by at least about 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, and up to at least about 200%.

6. PERSONAL CARE COMPOSITIONS 6.0.1 Personal Care Compositions Comprising Modified Variant BBPIs

The present invention provides personal care compositions comprising at least one modified variant BBPI for medical and nonmedical conditions. In some embodiments, the present invention provides personal care compositions and the methods of their use. In some embodiments, the invention provides personal care compositions for use in the skin care. In other embodiments, the personal care compositions are for use in hair care. In some embodiments, the personal care compositions for use in skin care include cosmetic compositions. In other embodiments, the personal care compositions of the invention are for use in the treatment of various disorders.

As described in greater detail herein, the present invention provides compositions for use in numerous aspects of personal care, including but not limited to hair and skin care, as well as cosmetics (e.g., make-up). For example, the present invention provides compositions that find use in daily personal care, skin care, sun care (e.g., sunscreens, as well as tanners), hair care (e.g., shampoos, leave-on and/or rinse off conditioners, hair tonics, hair sprays, gels, foams, mousses, setting products, hair colorants, permanent formulations, other styling and cleaning products, etc.), after-sun care for skin, hair and lips, oral care (e.g., toothpastes and gels, mouthwashes, rinses, etc.), bathing (e.g., washes, shower soaps, bath soaps, salts, pearls, etc.), skin lighteners, cleansing treatments for skin conditions (e.g., pimples, acne, skin toners, etc.), depilatories, wet wipes, deodorants, antiperspirants, facial masks, shaving (e.g., shaving creams, gels, etc.), after-shave, skin peeling (e.g., exfoliants), intimate care products (e.g., feminine hygiene products), personal fresheners, and foot care. The present invention also provides compositions that find use in cosmetics (e.g., foundations, mascara, eye shadows, eye liners, lipsticks, lip glosses, blushers, etc.). In addition, the present invention provides personal care compositions for use in ameliorating a hair condition associated with a disorder. In other embodiments, the personal care composition are for use in ameliorating a skin condition associated with a disorder. It is contemplated that the compositions of the present invention will find use various forms, including but not limited to solids, liquids, colloidal suspensions, emulsions, oils, gels, aerosols, foams, powders, pump sprays, etc., as well as being used in conjunction with items such as wet wipes, etc. Indeed, it is contemplated that the present invention will find use in any suitable form for the intended use(s).

In some embodiments, the personal care composition comprises a modified variant BBPI comprising a variant peptide and at least one amino acid substitution, as described in section 5.4.1 above. In other embodiments, the personal care composition comprises a modified variant BBPI comprising a variant peptide and a combination of amino acid substitutions, as described in section 5.4.2 above. In some embodiments, the personal care composition comprises a modified variant BBPI in which the equivalent chymotrypsin loop is a VEGF-binding peptide. In other embodiments, the personal care composition comprises a modified variant BBPI in which the equivalent chymotrypsin loop is an FGF5 binding peptide. In other embodiments, the personal care composition comprises a modified variant BBPI in which the equivalent chymotrypsin loop is a TGFβ binding peptide. In yet other embodiments, the personal care composition comprises a modified variant BBPI in which the equivalent chymotrypsin loop is a TNFα binding peptide. In some embodiments, the modified variant BBPI comprises from about 0.0001 weight percent to about 5 weight percent of the personal care composition, while in alternative embodiments, the modified variant BBPI comprises from about 0.001 weight percent to about 0.5 weight percent of the personal care composition, and in yet additional embodiments, the modified variant BBPI comprises from about 0.01 weight percent to about 1 weight percent of the personal care composition.

6.0.2 Personal Care Compositions Comprising Modified Variant VEGF-BBPIs

VEGF plays a central role in promoting angiogenesis as well as influencing diverse cell functions including cell survival, proliferation and the generation of nitric oxide and prostacyclin (Zachary et al., Cardiovasc Res, 49: 568-81 [2001]). The recognition of VEGF as a primary stimulus of angiogenesis in pathological conditions has led to various attempts to block VEGF activity. Inhibitory anti-VEGF receptor antibodies, soluble receptor constructs, antisense strategies, RNA aptamers against VEGF and low molecular weight VEGF receptor tyrosine kinase (RTK) inhibitors have all been proposed for use in interfering with VEGF signaling (See, Siemeister et al., [1998]). In fact, monoclonal antibodies against VEGF have been shown to inhibit human tumor xenograft growth and ascites formation in mice (See, Kim et al., [1993]; Asano et al., [1998]; Mesiano et al., [1998]; Luo et al., [1998a] and [1998b]; and Borgstrom et al., [1996] and [1998]).

Angiogenesis, involving VEGF and RTKs is not only involved in cancer development, as many other diseases or conditions affecting different physiological systems are angiogenesis-dependent, such as arthritis and atherosclerotic plaques (bone and ligaments), diabetic retinopathy, neovascular glaucoma, macular degeneration, ocular herpes, trachoma and corneal graft neovascularization (eye) and angiofibroma. VEGF expression is upregulated in the hyperplastic epidermis of psoriasis patients (Detmar and Yeo et al. [1995]), and in other skin diseases characterized by enhanced angiogenesis including, scleroderma, rosacea, hemangioma, contact dermatitis, and hypertrophic scarring of the skin. Targeted overexpression of VEGF in the epidermis of transgenic mice was reported to result in enhanced skin vascularization with equal numbers of tortuous and leaky blood vessels (See e.g., Brown et al., [1998]). Also, chronic synthesis of VEGF in mouse skin leads to the first histologically equivalent murine model of human psoriasis (Xia et al., [2003]) that is reversible by binding agents specific for VEGF. In addition, ultraviolet radiation induces VEGF production in keratinocytes and enhances cutaneous angiogenesis (Kim et al., Soc. Investigative Dermatol. 126:2697 [2006]; Blaudshun et al., FEBS Let. 474:195-200; Kosmadaki et al., FASEB J. 17:446-8 [2003]).

In addition, the expression of VEGF in the outer root sheath of murine hair follicles was found to be temporally and spatially associated with capillary proliferation during anagen. Transgenic overexpression of VEGF in the outer root sheath increased perifollicular vascularization and led to accelerated hair growth following depilation and the growth of larger hairs (Yano et al. J Clin Invest 107: 409-17 [2001]).

Thus, VEGF is involved in the vascularization associated with pathologic and non-pathologic conditions.

The invention provides for personal skin care and/or hair care compositions that comprise a modified variant VEGF-BBPI. The VEGF-BBPIs comprised in the personal care compositions of the invention are modified variant BBPIs in which the equivalent chymotrypsin loop of the precursor scaffold of the VEGF-BBPI has been replaced by a VEGF variant peptide, and which further comprise at least one amino acid substitution as described in sections 5.4.1 and 5.4.2. In some embodiments of the present invention, binding of the modified variant VEGF-BBPI to VEGF prevents VEGF from increasing perifollicular vascularization and inhibits the VEGF from promoting hair growth. In other embodiments, binding of the modified variant VEGF-BBPI to VEGF prevents VEGF from increasing vascularization of the skin in a subject suffering from an angiogenic skin disorder e.g. hemangioma and lichen planus. In yet other embodiments, binding of the modified variant VEGF-BBPI to VEGF prevents VEGF from promoting disregulated angiogenesis associated with inflammatory skin disorders including psoriasis, scleroderma, venous ulcers, acne, rosacea, warts, eczema, and lymphangiogenesis. However, it is not intended that the present invention be limited to any particular mechanism.

In some embodiments, the personal care composition comprises a VEGF-BBPI in which the chymotrypsin loop is a VEGF-binding peptide chosen from U.S. application Ser. Nos. 09/832,723 and 10/984,270, including peptides ACYNLYGWTC (SEQ ID NO:9), KYYLYWW (SEQ ID NO:458), TLWKSYW (SEQ ID NO:459), DLYWW (SEQ ID NO:460), SKHSQIT (SEQ ID NO:468) KTNPSGS (SEQ ID NO:469) RPTGHSL (SEQ ID NO:470), KHSAKAE (SEQ ID NO:471) KPSSASS (SEQ ID NO:472), PVTKRVH (SEQ ID NO:473), TLHWWVT (SEQ ID NO:492), PYKASFY (SEQ ID NO:493), PLRTSHT (SEQ ID NO:494), EATPROT (SEQ ID NO:495), NPLHTLS (SEQ ID NO:496), KHERIWS (SEQ ID NO:497), ATNPPPM (SEQ ID NO:498), STTSPNM (SEQ ID NO:499), ADRSFRY (SEQ ID NO:500), PKADSKQ (SEQ ID NO:501), PNQSHLH (SEQ ID NO:502), SGSETWM (SEQ ID NO:503), ALSAPYS (SEQ ID NO:504), KMPTSKV (SEQ ID NO:505), ITPKRPY (SEQ ID NO:506), KWIVSET (SEQ ID NO:507), PNANAPS (SEQ ID NO:508), NVQSLPL (SEQ ID NO:509), TLWPTFW (SEQ ID NO:510), NLWPHFW (SEQ ID NO:511), SLWPAFW (SEQ ID NO:512), SLWPHFW (SEQ ID NO:513), APWNSHI (SEQ ID NO:514), APWNLHI (SEQ ID NO:515), LPSWHLR (SEQ ID NO:516), PTILEWY (SEQ ID NO:517), TLYPQFW (SEQ ID NO:518), and HLAPSAV (SEQ ID NO:519). In some other embodiments, the VEGF variant sequences include, but are not limited to VEGF-binding peptides disclosed in U.S. application Ser. No. 11/919,717, including peptides KYYLSWW (SEQ ID NO:520), WYTLYKW (SEQ ID NO:521), TYRLYWW (SEQ ID NO:522), RYSLYYW (SEQ ID NO:523), YYLYYWK (SEQ ID NO:524), NYQLYGW (SEQ ID NO:525), TKWPSYW (SEQ ID NO:226), TLWKSYW (SEQ ID NO:527), PLWPSYW (SEQ ID NO:528), RLWPSYW (SEQ ID NO:529), TLWPKYW (SEQ ID NO:530), KYDLYWW (SEQ ID NO;531), RYDLYWW (SEQ ID NO:532), DYRLYWW (SEQ ID NO:533), DYKLYWW (SEQ ID NO:534), EYKLYWW (SEQ ID NO:535), and RYPLYWW (SEQ ID NO:536). In other embodiments, the VEGF binding peptide is chosen from SEQ ID NOS:9, 458, 459, 460, 468, 469, 470, 471, 472 and 473.

The scaffold in which the variant VEGF peptide is introduced to replace the chymotrypsin loop is chosen from the scaffolds of the soybean inhibitor from Glycine max (BBI; SEQ ID NO:13) or the mature and truncated form thereof (SEQ ID NO:185), the inhibitor from Dolichos biflorus (BBdb; SEQ ID NO:449), the soybean inhibitor D-II from Glycine max (BBsb3; SEQ ID NO:450), the inhibitor from Torresea (Amburana) cearensis (BBtc; SEQ ID NO:451), the BBI-AV scaffold of (SEQ ID NO:186), the BBIt-AV scaffold of (SEQ ID NO:187), the BBdb-AV scaffold of (SEQ ID NO:452), the BBsb3-AV scaffold of (SEQ ID NO:453), the BBtc-AV scaffold of (SEQ ID NO:454), the BBIt-VEGK scaffold of (SEQ ID NO:640), the BBIt-VEGT scaffold of (SEQ ID NO:641) and the BBIt-VEGKD scaffold of (SE ID NO:642). In addition, any wild-type BBPI precursor scaffolds, such as those disclosed by Prakash et al. (supra), may be used to generate variant BBPI scaffolds. In some embodiments, the scaffold of the VEGF-BBPI is that of SEQ ID NO:187.

In some embodiments, the backbone of the modified variant VEGF-BBPI comprises at least one amino acid substitution at least at one amino acid position chosen from positions equivalent to 1, 4, 5, 11, 13, 18, 25, 27, 29, 31, 38, 40, 50, 52, 55, and 65 of the variant BBI of SEQ ID NO:187, as described in section 5.4.1.

In other embodiments, the backbone of the modified variant VEGF-BBPI comprises a combination of amino acid substitutions chosen from a combination two, three, four, five, six, seven or eight amino acid substitutions as recited above in section 5.4.2.

In other embodiments, the backbone of the modified variant VEGF-BBPI comprises a combination of amino acid substitutions chosen from a combination of amino acid substitutions chosen from 13I-29P-50T-52A, 13I-40K-50T-52A, 13I-25K-29P-52K, 13I-29P-40K-50T-52A, 13I-25R-27A-29P-31A-50K-52T, 13I-25R-27A-29P-31A-40H-50K-52T, 13I-25K-27A-29R-31E-40K-50Q-52Q, 13I-25K-27A-29R-31A-40H-50R-52L.

In some embodiments, the personal care compositions of the invention comprise a modified variant BBPI in which the equivalent chymotrypsin loop of the precursor scaffold is replaced with a VEGF variant peptide chosen from SEQ ID NOS:9, 458, 459, 460, 468, 469, 470, 471, 472 and 473, wherein the scaffold is that of SEQ ID NO:187, and which comprises a combination of amino acid substitutions chosen from 13I-40K-50T-52A, 13I-25K-29P-52K, 13I-29P-40K-50T-52A, 13I-25R-27A-29P-31A-50K-52T, 13I-25R-27A-29P-31A-40H-50K-52T, 13I-25K-27A-29R-31E-40K-50Q-52Q, 13I-25K-27A-29R-31A-40H-50R-52L. In some embodiments, the personal care compositions comprise a VEGF-BBPI chosen from the VEGF-BBPIs of SEQ ID NOS:601, 602, 627, 628, 629, 630, 631, 643, 491, 632, 633, 634, 635 and 636.

In some embodiments, the invention provides a personal care composition comprising a VEGF-BBPI that binds to VEGF. In alternative embodiments, the binding of the VEGF-BBPI to VEGF blocks the downstream activity of VEGF. In some embodiments, the composition is capable of modulating angiogenesis.

In some embodiments, the personal care composition comprises a modified variant BBPI-VEGF for use in skin care. In some embodiments, the skin care compositions are for cosmetic use in improving the appearance of skin. In other embodiments, the skin care compositions are for therapeutic use in improving the appearance of skin in a subject suffering from a skin disorder. In some embodiments, the skin disorder is an angiogenic skin disorder. In additional embodiments, the skin disorder is at least one chosen from psoriasis, scleroderma, venous ulcers, acne, rosacea, warts, eczema, hemangiomas and lymphangiogenesis. In some embodiments, the skin disorder is rosacea. In other embodiments, the skin disorder is psoriasis.

In one embodiment, the personal skin care composition comprising a VEGF-BBPI is chosen from skin creams, lotions, sprays, emulsions, colloidal suspensions, foams, aerosols, liquids, gels, sera, and solids. In another embodiment, the personal care composition is a skin care composition selected from moisturizing body washes, body washes, antimicrobial cleansers, skin protective creams, body lotions, facial creams, moisturizing creams, facial cleansing emulsions, facial gels, facial sera, surfactant-based facial cleansers, facial exfoliating gels, anti-acne treatments, facial toners, exfoliating creams, facial masks, after shave balms, pre-shave balms, tanning compositions, skin lightening compositions, skin redness reduction compositions, sunscreens, depilatories, hair growth inhibitors, and radioprotectives. Radioprotectives are chosen from non-water-resistant sunscreens, very water-resistant sunscreens, and water-in-silicone sunscreens.

In one embodiment, the personal care skin care composition comprising a VEGF-BBPI is comprises topically applied over-the-counter compositions, anti-fungal treatments, anti-acne treatments, skin protectants, sunscreens, deodorants, and antiperspirants. In other embodiments, the skin care composition is capable of lightening skin tone, reducing redness, preventing skin tone darkening or preventing color development.

The present invention also provides personal care compositions that are cosmetic compositions. In some preferred embodiments, the cosmetic compositions comprising a VEGF-BBPI are chosen from pressed powder formulations and foundations. In some preferred embodiments, the cosmetic compositions comprise at least one pigment.

In yet additional embodiments, the makeup compositions are pressed powder formulations selected from loose powders, blushes, and bronzing powders. In still further embodiments, the makeup compositions are foundations selected from water-in-oil foundations, water-in-silicone foundations, oil-in-water foundations, anhydrous makeup sticks, and cream-to-powder foundations.

In some embodiments, the personal skin care compositions comprise a modified variant VEGF-BBPI, as set forth herein, and a physiologically acceptable carrier or excipient. Preferably, the VEGF-BBPI is present in an amount of about 0.0001% to about 5% by weight based on the total weight of the composition. Also preferably, the VEGF-BBPI is present in an amount of about 0.001% to about 0.5% by weight based on the total weight of the composition. The composition may be in the form of an emulsified vehicle, such as a nutrient cream or lotion, a stabilized gel or dispersion system, a treatment serum, a liposomal delivery system, a topical pack or mask, a surfactant-based cleansing system such as a shampoo or body wash, an aerosolized or sprayed dispersion or emulsion, a hair or skin conditioner, styling aid, or a pigmented product such as makeup, as well as other suitable make-up and cosmetic preparations. In some embodiments, the carrier is preferably at least one selected from the group consisting of water, propylene glycol, ethanol, propanol, glycerol, butylene glycol and polyethylene glycol.

In some other embodiments, the invention provides a personal care composition that comprises a modified variant VEGF-BBPI for use in hair care.

In some embodiments, the hair care compositions find use in inhibiting hair growth. In other embodiments, inhibition of hair growth comprises hair removal for treatment of at least one disease or condition for which decreased hair growth is desirable. In some embodiments, inhibition and/or removal comprises depilation. In some embodiments, the hair is selected from the group consisting of facial hair, leg hair, arm hair, and torso hair.

In one embodiment, the hair care composition is selected from the group consisting of shampoos, conditioners, hair styling compositions, hair colorants, permanent wave formulations, creams, gels, mousses, sprays, emulsions, colloidal suspensions, liquids, foams, and solids. In some embodiments, the hair care composition further comprises a radioprotective. As described for the personal skin care compositions the radioprotective is a sunscreen chosen from non-water-resistant sunscreens, very water-resistant sunscreens, and water-in-silicone sunscreens. In other embodiments, the radioprotective is a sunscreen chosen from non-water-resistant sunscreens, very water-resistant sunscreens, and water-in-silicone sunscreens.

In some embodiments, the personal hair care compositions comprise a modified variant VEGF-BBPI, as set forth herein, and a physiologically acceptable carrier or excipient. Preferably, the VEGF-BBPI is present in an amount of about 0.0001% to about 5% by weight based on the total weight of the composition. Also preferably, the VEGF-BBPI is present in an amount of about 0.001% to about 1% by weight based on the total weight of the composition. The composition may be in the form of an emulsified vehicle, such as a nutrient cream or lotion, a stabilized gel or dispersion system, a treatment serum, a liposomal delivery system, a topical pack or mask, a surfactant-based cleansing system such as a shampoo or body wash, an aerosolized or sprayed dispersion or emulsion, a hair or skin conditioner, styling aid, or a pigmented product such as makeup, as well as other suitable make-up and cosmetic preparations. In some embodiments, the carrier is preferably at least one selected from the group consisting of water, propylene glycol, ethanol, propanol, glycerol, butylene glycol and polyethylene glycol.

In other embodiments, the personal care compositions of the invention are for use in the treatment of various diseases associated with elevated levels of VEGF.

The present invention also provides methods for inhibiting hair growth of a subject, comprising the steps of providing the personal care VEGF hair care composition of the present invention; providing a subject to be treated; and applying the composition to the subject in an area in which inhibition of hair growth is desired. In some embodiments, the inhibition of hair growth comprises inhibiting the growth of the subject's hair, wherein the hair to be inhibited is selected from the group consisting of facial air, underarm hair, leg hair, torso hair, and arm hair, and head hair. In additional embodiments, the method for inhibiting hair growth using a VEGF hair care composition of the invention comprises a personal care composition comprising a VEGF-BBPI having an amino acid sequence chosen from SEQ ID NOS SEQ ID NOS:601, 602, 627, 628, 629, 630, 631, 643, 491, 632, 633, 634, 635 and 636.

In another embodiment, the invention provides a method for improving the appearance and/or condition of skin in a subject suffering from a skin disorder, comprising providing the personal care composition of the present invention; providing a subject to be treated; and applying the composition to the affected skin of the subject. In some embodiments, the skin disorder is chosen from psoriasis, venous ulcers, acne, rosacea, warts, eczema, hemangiomas, cutaneous lichen planus, and lymphangiogenesis, etc. In some particularly preferred embodiments, the skin disorder is rosacea. In other embodiments, the skin disorder is psoriasis. In additional embodiments, the method for improving the appearance and/or condition of skin in a subject suffering from a skin disorder using a VEGF skin care composition of the invention comprises a personal care composition comprising a VEGF-BBPI having an amino acid sequence chosen from SEQ ID NOS:601, 602, 627, 628, 629, 630, 631, 643, 491, 632, 633, 634, 635 and 636.

6.0.3 Personal Care Compositions Comprising Modified Variant FGF-5-BBPIs

The Fibroblast Growth Factor (FGF) family is a superfamily of growth factors containing at least 23 members, many of which are potent regulators of cell proliferation, differentiation and cell function. All of the FGFs have a conserved 120 amino acid core. Members of the family share conserved cysteine residues and 30-50% sequence homology at the amino acid level. The molecular weight of the FGFs ranges from 7 kDa for FGF-1 to 38 kDa for FGF-5. The length of the proteins is from 60 amino acids in the case of an FGF-1 splice variant to 288 amino acids for FGF-2. Binding to heparin is an essential step required for an FGF factor to interact with cell surface receptors. FGF5 is a secreted signaling protein consisting of 268 amino acids with a 17 amino acid signal sequence and a 251 amino acid mature peptide. The human gene also gives rise to a glycosylated alternate splice form that is 18 kDa in size and 123 amino acid in length. The murine homologue of FGF-5 was cloned and found to be 84% homologous to the human protein at the amino acid sequence level. Human FGF-5 consists of three exons and maps to chromosome 4q21 and cross-reacts with murine FGF-5.

Formation of hair follicles involves a complex series of steps: growth (anagen), regression (catagen), rest (telogen) and shedding (exogen). FGF-5 has been implicated as one of the major drivers of the transition from anagen to catagen in the hair cycle. Expression of FGF-5 is detected in hair follicles from wild-type mice and is localized to the outer root sheath during the anagen phase. Mice homozygous for a predicted null allele of FGF-5, fgf5neo, have abnormally long hair (See, Hebert et al., Cell 78: 1017-25 [1994]). The phenotype appears identical to that of mice homozygous for the spontaneous mutation angora (go). Recently, partial FGF-5 sequences, FGF5S, thought to compete with FGF-5 in binding to the receptor have been identified, (See, Ito et al., J. Cell Physiol., 197:272-83 [2003]).

The invention provides personal hair care compositions that comprises at least one modified variant FGF-BBPI for use in promoting hair growth and/or preventing hair loss. In one embodiment, the invention provides FGF-BBPI compositions for personal skin care. In other embodiments, the invention provides FGF-BBPI compositions for hair care. The FGF-BBPIs comprised in the personal care compositions of the invention are modified variant BBPIs in which the equivalent chymotrypsin loop of the precursor scaffold of the FGF-5-BBPI has been replaced by an FGF-5 variant peptide, and which further comprise at least one amino acid substitution as described in sections 5.4.1 and 5.4.2. In the present invention, binding of the modified variant FGF-BBPI to FGF-5 prevents FGF-5 from interacting with its cognate receptor and inhibits transition from the anagen to the catagen promoting hair growth and preventing hair loss. However, it is not intended that the present invention be limited to any particular mechanism.

In some embodiments, personal care composition comprises at least one FGF-BBPI in which the chymotrypsin loop is an FGF-5 variant peptide chosen from CACRTQPYPLCF (MM007; SEQ ID NO:430), CICTWIDSTPC (PS2; SEQ ID NO:431), CYGLPFTRC (SEQ ID NO:537), CEEIWTMLC (SEQ ID NO:538), CWALTVKTC (SEQ ID NO:539), CLTVLWTTC (SEQ ID NO:540), CTLWNRSPC (SEQ ID NO:541), CHYLLTNYC (SEQ ID NO:542), CRIHLAHKC (SEQ ID NO:543), TNIDSTP (SEQ ID NO:544), HLQTTET (SEQ ID NO:545), SLNNLTV (SEQ ID NO:546), TNIDSTP (SEQ ID NO:547), TNIDSTP (SEQ ID NO:548), LRILANK (SEQ ID NO:549), LLTPTLN (SEQ ID NO:550), ALPTHSN (SEQ ID NO:551), TNIDSTP (SEQ ID NO:552), LCRRFEN (SEQ ID NO:553), TNIDSTP (SEQ ID NO:554), TNIDSTP (SEQ ID NO:555), HLQTTET (SEQ ID NO:556), PLGLCPP (SEQ ID NO:557), GYFIPSI (SEQ ID NO:558), TKIDSTP (SEQ ID NO:559), HLQTTET (SEQ ID NO:560), WNIDSTP (SEQ ID NO:561), TWIDWTP (SEQ ID NO:562), RTQPYPL (SEQ ID NO:670) and TWIDSTP (SEQ ID NO:671). In other embodiments, the FGF variant peptide is chosen from SEQ ID NOS: SEQ ID NOS:430, 431, 670 and 671.

The scaffold in which the variant FGF peptide is introduced to replace the chymotrypsin loop is chosen from the scaffolds of the soybean inhibitor from Glycine max (BBI; SEQ ID NO:13) or the mature and truncated form thereof (SEQ ID NO:185), the inhibitor from Dolichos biflorus (BBdb; SEQ ID NO:449), the soybean inhibitor D-II from Glycine max (BBsb3; SEQ ID NO:450), the inhibitor from Torresea (Amburana) cearensis (BBtc; SEQ ID NO:451), the BBI-AV scaffold of (SEQ ID NO:186), the BBIt-AV scaffold of (SEQ ID NO:187), the BBdb-AV scaffold of (SEQ ID NO:452), the BBsb3-AV scaffold of (SEQ ID NO:453), the BBtc-AV scaffold of (SEQ ID NO:454), the BBIt-VEGK scaffold of (SEQ ID NO:640), the BBIt-VEGT scaffold of (SEQ ID NO:641) and the BBIt-VEGKD scaffold of (SE ID NO:642). In addition, any wild-type BBPI precursor scaffolds, such as those disclosed by Prakash et al. (supra), may be used to generate variant BBPI scaffolds. In some embodiments, the scaffold of the VEGF-BBPI is that of SEQ ID NO:187.

In some embodiments, the backbone of the modified variant FGF-BBPI comprises at least one amino acid substitution at least at one amino acid position chosen from positions equivalent to 1, 4, 5, 11, 13, 18, 25, 27, 29, 31, 38, 40, 50, 52, 55, and 65 of the variant BBI of SEQ ID NO:187, as recited in section 5.4.1. In other embodiments, the backbone of the modified variant FGF-BBPI comprises a combination of amino acid substitutions chosen from a combination two, three, four, five, six, seven or eight amino acid substitutions as recited above in section 5.4.2. In other embodiments, the backbone of the modified variant FGF-BBPI comprises a combination of amino acid substitutions chosen from a combination of amino acid substitutions chosen from 13I-29P-50T-52A, 13I-40K-50T-52A, 13I-25K-29P-52K, 13I-29P-40K-50T-52A, 13I-25R-27A-29P-31A-50K-52T, 13I-25R-27A-29P-31A-40H-50K-52T, 13I-25K-27A-29R-31E-40K-50Q-52Q, 13I-25K-27A-29R-31A-40H-50R-52L. In some embodiments, the combination of substitutions is 13I-29P-50T-52A.

In some embodiments, the personal care compositions of the invention comprise an FGF-BBPI in which the equivalent chymotrypsin loop of the precursor scaffold is replaced with an FGF variant peptide chosen from SEQ ID NOS:433 and 434, wherein the scaffold is that of SEQ ID NO:187, and which comprises the combination of amino acid substitutions 13I-40K-50T-52A. Thus, in some embodiments, the personal care compositions comprise a FGF-BBPI chosen from the FGF-BBPIs of SEQ ID NOS:439 and 441.

In some embodiments, the invention provides a personal care composition comprising a FGF-BBPI that binds to FGF. In alternative embodiments, the binding of the FGF-BBPI to FGF blocks the downstream activity of FGF. In some embodiments, the composition is capable of promoting hair growth.

In some embodiments, the personal care composition comprising a modified variant BBPI-FGF is for use in skin care. In some embodiments, the skin care compositions are cosmetic compositions for use in promoting hair growth. In some embodiments, the skin care compositions are for use in promoting hair growth in a subject suffering from a disease or condition that involves hair loss. In some of these embodiments, the disease or condition is at least one selected from the group consisting of inflammatory alopecias, pseudopelade, scleroderma, tick bites, lichen planus, psoriasis, lupus, seborrheic dermatitis, loose hair syndrome, hemochromatosis, androgenic alopecia, alopecia areata, cancer, conditions that affect defective hair fiber production, and environmental factors that affect hair production. In a preferred embodiment, the disease is androgenic alopecia or alopecia areata.

In one embodiment, the personal care composition comprising an FGF-BBPI is a skin care composition is chosen from skin creams, lotions, sprays, emulsions, colloidal suspensions, foams, aerosols, liquids, gels, sera, and solids. In another embodiment, the personal care composition is a skin care composition selected from moisturizing body washes, body washes, antimicrobial cleansers, skin protective creams, body lotions, facial creams, moisturizing creams, facial cleansing emulsions, facial gels, facial sera, surfactant-based facial cleansers, facial exfoliating gels, anti-acne treatments, facial toners, exfoliating creams, facial masks, after shave balms, pre-shave balms, tanning compositions, skin lightening compositions, skin redness reduction compositions, sunscreens, depilatories, hair growth inhibitors, and radioprotectives. Radioprotectives are chosen from non-water-resistant sunscreens, very water-resistant sunscreens, and water-in-silicone sunscreens.

In one embodiment, the personal skin care composition comprising an FGF-BBPI is a skin care composition comprising topically applied over-the-counter compositions, anti-fungal treatments, anti-acne treatments, skin protectants, sunscreens, deodorants, and antiperspirants.

In some embodiments, the personal skin care compositions comprise a modified variant VEGF-BBPI, as set forth herein, and a physiologically acceptable carrier or excipient. Preferably, the VEGF-BBPI is present in an amount of about 0.0001% to about 5% by weight based on the total weight of the composition. Also preferably, the VEGF-BBPI is present in an amount of about 0.001% to about 0.5% by weight based on the total weight of the composition. The composition may be in the form of an emulsified vehicle, such as a nutrient cream or lotion, a stabilized gel or dispersion system, a treatment serum, a liposomal delivery system, a topical pack or mask, a surfactant-based cleansing system such as a shampoo or body wash, an aerosolized or sprayed dispersion or emulsion, a hair or skin conditioner, styling aid, or a pigmented product such as makeup, as well as other suitable make-up and cosmetic preparations. In some embodiments, the carrier is preferably at least one selected from the group consisting of water, propylene glycol, ethanol, propanol, glycerol, butylene glycol and polyethylene glycol.

In other embodiments, the invention provides a personal care composition comprising a modified variant FGF-BBPI for use in hair care.

In some embodiments, the hair care compositions find use in promoting hair growth. In other embodiments, modulation comprises promoting hair growth in a subject suffering from a disease or condition that involves hair loss. In some of these embodiments, the disease or condition is at least one selected from the group consisting of inflammatory alopecias, pseudopelade, scleroderma, tick bites, lichen planus, psoriasis, lupus, seborrheic dermatitis, loose hair syndrome, hemochromatosis, androgenic alopecia, alopecia areata, cancer, conditions that affect defective hair fiber production, and environmental factors that affect hair production. In a preferred embodiment, the disease is androgenic alopecia or alopecia areata.

In one embodiment, the hair care composition is selected from the group consisting of shampoos, conditioners, hair styling compositions, hair colorants, permanent wave formulations, creams, gels, mousses, sprays, emulsions, colloidal suspensions, liquids, foams, and solids. In some embodiments, the hair care composition further comprises a radioprotective. As described for the personal skin care compositions the radioprotective is a sunscreen chosen from non-water-resistant sunscreens, very water-resistant sunscreens, and water-in-silicone sunscreens. In other embodiments, the radioprotective is a sunscreen chosen from non-water-resistant sunscreens, very water-resistant sunscreens, and water-in-silicone sunscreens.

In some embodiments, the personal hair care compositions comprise a modified variant FGF-BBPI, as set forth herein, and a physiologically acceptable carrier or excipient. Preferably, the FGF-BBPI is present in an amount of about 0.0001% to about 5% by weight based on the total weight of the composition. Also preferably, the FGF-BBPI is present in an amount of about 0.001% to about 0.5% by weight based on the total weight of the composition. The composition may be in the form of an emulsified vehicle, such as a nutrient cream or lotion, a stabilized gel or dispersion system, a treatment serum, a liposomal delivery system, a topical pack or mask, a surfactant-based cleansing system such as a shampoo or body wash, an aerosolized or sprayed dispersion or emulsion, a hair or skin conditioner, styling aid, or a pigmented product such as makeup, as well as other suitable make-up and cosmetic preparations. In some embodiments, the carrier is preferably at least one selected from the group consisting of water, propylene glycol, ethanol, propanol, glycerol, butylene glycol and polyethylene glycol.

In other embodiments, the personal care FGF-BBPI compositions of the invention are for use in the treatment of various diseases associated with elevated levels of FGF.

The present invention also provides methods for promoting hair growth of a subject, comprising the steps of providing the personal care FGF composition of the present invention; providing a subject to be treated; and applying the composition to the subject in an area in which promotion of hair growth is desired. In some embodiments, the FGF composition is a skin care composition. In other embodiments, the FGF composition is a hair composition. In some embodiments, promotion of hair growth comprises promoting the growth of the subject's hair, wherein the hair growth to be promoted is chosen from facial air, underarm hair, leg hair, torso hair, and arm hair, and head hair. In additional embodiments, the method for promoting hair growth using an FGF hair care composition of the invention comprises applying a personal care composition comprising an FGF-BBPI having an amino acid sequence chosen from SEQ ID NOS:439 and 441.

6.0.4 Personal Care Compositions Comprising Modified Variant TGFβ-BBPIs

Proteins of the Transforming Growth Factor-β (TGFβ) family are synthesized by almost all cells. The TGFβs are a group of stable, multifunctional polypeptide growth factors whose activities include, among other things, context-specific inhibition and stimulation of cell proliferation, control of the extracellular matrix, degradation and control of mesenchymal-epithelial interactions during embryogenesis, mediation of cell and tissue responses to injury, control of carcinogenesis and modulation of immune responses. TGFβ-1 is synthesized by virtually all cells (with only a few exceptions). TGFβ-1 has been found in the highest concentration in human platelets and mammalian bone. TGFβ-1 has many functions including suppression of cell proliferation, enhancement of extracellular matrix deposition and physiological immunosuppression. TGFβ-1 has also been determined to be biologically active in hair follicle development. Human TGFβ-1 is a 25.0 kDa protein with subunits that contain approximately 112 amino acids per subunit. Two different receptor proteins are involved in TGFβ-1 binding and signaling, namely TGF-RβII and TGF-RβI. TGFβ-2 is expressed in a variety of cells, including keratinocytes, fibroblasts, osteoclasts, thymic epithelium, skeletal muscle cells, prostatic epithelium, bronchial epithelium, neurons and astrocytes, visceral smooth muscle, macrophages and various other cells. TGFβ-2 has many fundamental activities, including function as a growth inhibitor for most cells, an enhancer for deposition of the extracellular matrix, and immunosuppression. The mature region is 71% identical to TGFβ-1, 80% identical to TGFβ-3, and 97% identical to the mouse homologue of the same protein at the amino acid level. TGFβ-2 dimerizes with formation of disulfide bonds between the ‘pro’ regions and disulfide bonds between the mature regions. TGFβ-2 is synthesized as a pre-procytokine with a 19 amino acid signal sequence, a 283 pro-region and a 112 mature amino acid segment. The receptor for TGFβ-2 forms a heterotetrameric complex of two type I signal-transduction receptors and two type II ligand-binding receptors.

Formation of hair follicles involves a complex series of steps: growth (anagen), regression (catagen), rest (telogen) and shedding (exogen) (See, Sten and Paus, Physiol. Rev, Exp. Dermatol., 8:229-233 [1999]). TGFβs have been implicated as one of the major drivers of the transition from anagen to catagen in the hair cycle (See e.g., Foitzik et al, FASEB J., 5:752-760 [2000]; and Soma et al. J. Infect. Dis., J. Invest. Dermatol., 118:993-9997 [2002]), and TGFβ2 is both a required and sufficient inducer of murine hair follicle morphogenesis (See, Foitzik et al., Develop. Biol., 212:278-289 [1999]). Conditional TGFβ-1 expression in transgenic mice demonstrates that one can induce alopecia reversibly (See, Liu et al., Proc. Natl. Acad. Sci. USA 98:9139-9144 [2001]). In addition, TGFβ-1 mutants have been associated with the delay of catagen onset in mice (See, Foitzik et al, [2000], supra). Recently, it has been shown that catagen can be delayed through the use of TGFβ-2 antibodies (See, Soma et al., [2002], supra). Finally, androgens that induce TGFβ-1 production in balding dermal papilla cells can inhibit epithelial cell growth (Inui et al., FASEB J., 14:1967-1969 [2002]).

TGFβ is also a potent stimulus of connective tissue accumulation, and is implicated in the pathogenesis of scleroderma and other fibrotic disorders (Blobe et al., N Engl J Med 342:1350-1358 [2000]). Scleroderma is a chronic autoimmune disease characterized by early inflammation and vascular injury, followed by progressive fibrosis of the skin and other organs (Kissin and Korn, Rheum Dis Clin North Am 29:351-369 [2003]). The most evident symptom is usually the hardening of the skin and associated scarring.

TGFβ has also been implicated in the formation of skin tumors (Li et al., Molecular Carcinogenesis 45:389-396 [2006]). The TGFβ signaling pathway is one of the most important mechanisms in the maintenance of epithelial homeostasis. Alterations leading to either the repression or enhancement of this pathway have been shown to affect cancer development. Although TGFβ inhibits growth of normal epithelial cells, it is paradoxically overexpressed in many epithelial cancers. It has been postulated that TGFβ acts as a tumor suppressor at the early stages of carcinogenesis, but overexpression of TGFβ at late stages of carcinogenesis may be a critical factor for tumor invasion and metastasis.

The invention provides personal care compositions that comprise a modified variant TGF-BBPI. The TGFβ-BBPI comprised in the personal care compositions of the invention is a modified variant TGFβ-BBPI in which the chymotrypsin loop of the precursor scaffold of the TGF-BBPI has been replaced by a TGFβ variant peptide, and which further comprise at least one amino acid substitution as described in sections 5.4.1 and 5.4.2. In some embodiments, binding of the modified variant TGF-BBPI to TGFβ1 and/or TGFβ2 prevents TGFβ1 and/or TGFβ2 from interacting with its cognate receptor and inhibits transition from the anagen to the catagen to promote hair growth and preventing hair loss. In other embodiments, binding of the modified variant TGF-BBPI to TGFβ1 and/or TGFβ2 prevents TGFβ1 and/or TGFβ2 from interacting with its cognate receptor to prevent fibrosis of the skin that is characteristic of scleroderma. In other embodiments, binding of the modified variant TGF-BBPI to TGFβ1 and/or TGFβ2 prevents TGFβ1 and/or TGFβ2 from interacting with its cognate receptor to prevent progression of benign to malignant skin lesions. However, it is not intended that the present invention be limited to any particular mechanism.

In some embodiments, the TGFβ variant peptide comprised in the TGFβ-BBPI is chosen from CLCPENINVLPCN (PEN3; SEQ ID NO:436), CICKHNVDWLCF (MMO21W; SEQ ID NO:437), CICWTQHIHNCF (WTQ; SEQ ID NO:438), CVTTDWIEC (SEQ ID NO:563), CYYSQFHQC (SEQ ID NO:564), CPTLWTHMC (SEQ ID NO:565), QSACIVYYVGRKPKVECASSD (SEQ ID NO:566), QSACILYYIGKTPKIECASSD (SEQ ID NO:567), QSACILYYVGRTPKVECASSD (SEQ ID NO:568), acetyl-LCPENDNVSPCY-cohn2 (SEQ ID NO:569), KHNVRLL (SEQ ID NO:570), NDTPSYF (SEQ ID NO:571), AKLYAGS (SEQ ID NO:572), RGPAHSL (SEQ ID NO:573), NSLAERR (SEQ ID NO:574), HPLASPH (SEQ ID NO:575), QPWNKLK (SEQ ID NO:576), AWLr/Mipy (SEQ ID NO:577), PTKPAQQ (SEQ ID NO:578), PSLNRPQ (SEQ ID NO:579), HHARQEW (SEQ ID NO:580), RHHTPGP (SEQ ID NO:581), ASAINPH (SEQ ID NO:582), CHGYDRAPC (SEQ ID NO:644), CFAPADQAC (SEQ ID NO:645), CIPSRFITC (SEQ ID NO:646), CHGHTKLAC (SEQ ID NO:647), CNGKSKLAC (SEQ ID NO:648), PENINVLP (SEQ ID NO;672), KHNVDWL (SEQ ID NO:673), and WTQHIHNC (SEQ ID NO:674). SEQ ID NOS:644-648 are TGFβ1-binding peptides, while SEQ ID NOS:463, 437, 438, 563-582 are TGFβ2-binding peptides.

In other embodiments, the TGFβ variant peptide is chosen from SEQ ID NOS: SEQ ID NOS:436, 437 and 438.

The scaffold in which the variant TGF peptide is introduced to replace the equivalent chymotrypsin loop is chosen from the scaffolds of the soybean inhibitor from Glycine max (BBI; SEQ ID NO:13) or the mature and truncated form thereof (SEQ ID NO:185), the inhibitor from Dolichos biflorus (BBdb; SEQ ID NO:449), the soybean inhibitor D-II from Glycine max (BBsb3; SEQ ID NO:450), the inhibitor from Torresea (Amburana) cearensis (BBtc; SEQ ID NO:451), the BBI-AV scaffold of (SEQ ID NO:186), the BBIt-AV scaffold of (SEQ ID NO:187), the BBdb-AV scaffold of (SEQ ID NO:452), the BBsb3-AV scaffold of (SEQ ID NO:453), the BBtc-AV scaffold of (SEQ ID NO:454), the BBIt-VEGK scaffold of (SEQ ID NO:640), the BBIt-VEGT scaffold of (SEQ ID NO:641) and the BBIt-VEGKD scaffold of (SE ID NO:642). In addition, any wild-type BBPI precursor scaffolds, such as those disclosed by Prakash et al. (supra), may be used to generate variant BBPI scaffolds. In some embodiments, the scaffold of the VEGF-BBPI is that of SEQ ID NO:187.

In some embodiments, the backbone of the modified variant TGFβ-BBPI comprises at least one amino acid substitution at least at one amino acid position chosen from positions equivalent to 1, 4, 5, 11, 13, 18, 25, 27, 29, 31, 38, 40, 50, 52, 55, and 65 of the variant BBI of SEQ ID NO:187, as recited in section 5.4.1.

In other embodiments, the backbone of the modified variant TGFβ-BBPI comprises a combination of amino acid substitutions chosen from a combination two, three, four, five, six, seven or eight amino acid substitutions as recited above in section 5.4.2.

In other embodiments, the backbone of the modified variant TGFβ-BBPI comprises a combination of amino acid substitutions chosen from a combination of amino acid substitutions chosen from 13I-29P-50T-52A, 13I-40K-50T-52A, 13I-25K-29P-52K, 13I-29P-40K-50T-52A, 13I-25R-27A-29P-31A-50K-52T, 13I-25R-27A-29P-31A-40H-50K-52T, 13I-25K-27A-29R-31E-40K-50Q-52Q, 13I-25K-27A-29R-31A-40H-50R-52L. In some embodiments, the combination of substitutions is 13I-29P-50T-52A. In some embodiments, the personal care compositions of the invention comprise a BBPI in which the equivalent chymotrypsin loop of the precursor scaffold is replaced with a TGFβ variant peptide chosen from SEQ ID NOS: 436, 437, 438, 672, 673, and 674, wherein the scaffold is that of SEQ ID NO:187, and which comprises the combination of amino acid substitutions 13I-29P-50T-52A. In some embodiments, the personal care compositions comprise a FGF-BBPI chosen from the TGFβ-BBPIs of SEQ ID NOS:443, 445 and 447.

In some embodiments, the invention provides a personal care composition comprising a TGFβ-BBPI that binds to TGFβ1 and/or TGFβ2. In alternative embodiments, the binding of the TGFβ-BBPI to TGFβ blocks the downstream activity of TGFβ. In some embodiments, the composition is capable of promoting hair growth.

In some embodiments, the personal care composition comprising a modified variant TGF-BBPI is for use in skin care. In some embodiments, the skin care compositions are cosmetic compositions for use in promoting hair growth.

In some embodiments, the skin care compositions are for use in promoting hair growth in a subject suffering from a disease or condition that involves hair loss. In some of these embodiments, the disease or condition is at least one selected from the group consisting of inflammatory alopecias, pseudopelade, scleroderma, tick bites, lichen planus, psoriasis, lupus, seborrheic dermatitis, loose hair syndrome, hemochromatosis, androgenic alopecia, alopecia areata, cancer, conditions that affect defective hair fiber production, and environmental factors that affect hair production. In a preferred embodiment, the disease is androgenic alopecia or alopecia areata.

In other embodiments, the personal care composition comprising a modified variant TGF-BBPI is for use in improving the appearance and/or condition of the skin of a subject suffering from scleroderma. In yet other embodiments, the personal care composition comprising a modified variant TGF-BBPI is for use in improving the appearance and/or condition of the skin of a subject suffering from skin cancer.

In one embodiment, the personal care composition comprising a TGFβ-BBPI is a skin care composition is chosen from skin creams, lotions, sprays, emulsions, colloidal suspensions, foams, aerosols, liquids, gels, sera, and solids. In another embodiment, the personal care composition is a skin care composition selected from moisturizing body washes, body washes, antimicrobial cleansers, skin protective creams, body lotions, facial creams, moisturizing creams, facial cleansing emulsions, facial gels, facial sera, surfactant-based facial cleansers, facial exfoliating gels, anti-acne treatments, facial toners, exfoliating creams, facial masks, after shave balms, pre-shave balms, tanning compositions, skin lightening compositions, skin redness reduction compositions, sunscreens, depilatories, hair growth inhibitors, and radioprotectives. Radioprotectives are chosen from non-water-resistant sunscreens, very water-resistant sunscreens, and water-in-silicone sunscreens.

In one embodiment, the personal care composition comprising a TGFβ-BBPI is a skin care composition comprising topically applied over-the-counter compositions, anti-fungal treatments, anti-acne treatments, skin protectants, sunscreens, deodorants, and antiperspirants.

The present invention also provides personal care compositions that are cosmetic compositions. In some preferred embodiments, the cosmetic compositions are selected from mascaras, pressed powder formulations, and foundations. In some preferred embodiments, the makeup compositions comprise at least one pigment.

In some preferred embodiments, the makeup composition comprising at least one pigment is a mascara selected from non-waterproof mascaras, waterproof mascaras, volumizing mascaras, lengthening mascaras, curling mascaras, anhydrous waterproof mascaras, water-based mascaras, and eyelash or eyebrow treatments.

In yet additional embodiments, the makeup compositions are pressed powder formulations selected from loose powders, blushes, eye shadows, and bronzing powders. In still further embodiments, the makeup compositions are foundations selected from water-in-oil foundations, water-in-silicone foundations, oil-in-water foundations, anhydrous makeup sticks, and cream-to-powder foundations.

In some embodiments, the personal skin care compositions comprise a modified variant TGF-BBPI, as set forth herein, and a physiologically acceptable carrier or excipient. Preferably, the TGF-BBPI is present in an amount of about 0.0001% to about 5% by weight based on the total weight of the composition. Also preferably, the TGF-BBPI is present in an amount of about 0.001% to about 0.5% by weight based on the total weight of the composition. The composition may be in the form of an emulsified vehicle, such as a nutrient cream or lotion, a stabilized gel or dispersion system, a treatment serum, a liposomal delivery system, a topical pack or mask, a surfactant-based cleansing system such as a shampoo or body wash, an aerosolized or sprayed dispersion or emulsion, a hair or skin conditioner, styling aid, or a pigmented product such as makeup, as well as other suitable make-up and cosmetic preparations. In some embodiments, the carrier is preferably at least one selected from the group consisting of water, propylene glycol, ethanol, propanol, glycerol, butylene glycol and polyethylene glycol.

In other embodiments, the invention provides a personal care composition that comprises a modified variant TGF-BBPI for use in hair care. In some embodiments, the hair care composition is for use in promoting hair growth.

In some embodiments, the hair care compositions find use in promoting hair growth. In other embodiments, modulation comprises promoting hair growth in a subject suffering from a disease or condition that involves hair loss. In some of these embodiments, the disease or condition is at least one selected from the group consisting of inflammatory alopecias, pseudopelade, scleroderma, tick bites, lichen planus, psoriasis, lupus, seborrheic dermatitis, loose hair syndrome, hemochromatosis, androgenic alopecia, alopecia areata, cancer, conditions that affect defective hair fiber production, and environmental factors that affect hair production. In a preferred embodiment, the disease is androgenic alopecia or alopecia areata.

In one embodiment, the hair care composition is selected from the group consisting of shampoos, conditioners, hair styling compositions, hair colorants, permanent wave formulations, creams, gels, mousses, sprays, emulsions, colloidal suspensions, liquids, foams, and solids. In some embodiments, the hair care composition further comprises a radioprotective. As described for the personal skin care compositions the radioprotective is a sunscreen chosen from non-water-resistant sunscreens, very water-resistant sunscreens, and water-in-silicone sunscreens. In other embodiments, the radioprotective is a sunscreen chosen from non-water-resistant sunscreens, very water-resistant sunscreens, and water-in-silicone sunscreens.

In some embodiments, the personal hair care compositions comprise a modified variant TGFβ-BBPI, as set forth herein, and a physiologically acceptable carrier or excipient. Preferably, the TGFβ-BBPI is present in an amount of about 0.0001% to about 5% by weight based on the total weight of the composition. Also preferably, the TGFβ-BBPI is present in an amount of about 0.001% to about 0.5% by weight based on the total weight of the composition. The composition may be in the form of an emulsified vehicle, such as a nutrient cream or lotion, a stabilized gel or dispersion system, a treatment serum, a liposomal delivery system, a topical pack or mask, a surfactant-based cleansing system such as a shampoo or body wash, an aerosolized or sprayed dispersion or emulsion, a hair or skin conditioner, styling aid, or a pigmented product such as makeup, as well as other suitable make-up and cosmetic preparations. In some embodiments, the carrier is preferably at least one selected from the group consisting of water, propylene glycol, ethanol, propanol, glycerol, butylene glycol and polyethylene glycol.

In other embodiments, the personal care TGF-BBPI compositions of the invention are for use in the treatment of various diseases associated with elevated levels of TGFβ1 and/or TGFβ2.

The present invention also provides methods for promoting hair growth of a subject, comprising the steps of providing the personal care TGF composition of the present invention; providing a subject to be treated; and applying the composition to the subject in an area in which promotion of hair growth is desired. In some embodiments, the TGF composition is a skin care composition. In other embodiments, the FGF composition is a hair composition. In some embodiments, promotion of hair growth comprises promoting the growth of the subject's hair, wherein the hair growth to be promoted is chosen from facial air, underarm hair, leg hair, torso hair, and arm hair, and head hair. In additional embodiments, the method for promoting hair growth using an TGF hair care composition of the invention comprises applying a personal care composition comprising a TGF-BBPI having an amino acid sequence chosen from SEQ ID NOS:443, 445 and 447.

In another embodiment, the invention provides a method for improving the appearance and/or condition of skin in a subject suffering from a skin disorder, comprising providing the personal skin care composition of the present invention; providing a subject to be treated; and applying the composition to the affected skin of the subject. In some embodiments, the skin disorder is chosen from psoriasis, scleroderma and skin cancer. In additional embodiments, the method for improving the appearance and/or condition of skin in a subject suffering from a skin disorder using a TGF skin care composition of the invention comprises a personal care composition comprising a TGF-BBPI having an amino acid sequence chosen from SEQ ID NOS: 443, 445 and 447.

6.0.5 Personal Care Compositions Comprising Modified Variant TNFα-BBPIs

Proteins of the Tissue Necrosis Factor (TNF) family are members of a large cytokine family. Tissue Necrosis Factor alpha (TNFα) is a prototypic member of the TNF family of ligands. It is produced by various immune cells, and plays a key role in immune-mediated inflammatory diseases.

TNFα is synthesized by T-lymphocytes, B cells, synoviocytes, fibroblasts, and macrophages initially as a 26 kD protein. Later it is cleaved by TNFα converting enzyme (the metalloproteinase, ADAMS 17) into a monomeric 17-kD molecule. Three of these molecules form, under physiologic conditions, a non-covalently bound, cone-shaped homotrimer that cross-links membrane-bound receptors that exist in 2 isoforms: TNF receptor I (TNF-RI) and TNF receptor II (TNF-RII). Its natural function is to stimulate the recruitment of neutrophils and monocytes to sites of infection and to activate these cells to eradicate microbes, exerting its function by binding to its corresponding TNF receptor on the surface of numerous cell types. It activates the vascular endothelium locally, causing vasodilation and increased permeability. Vascular endothelium adhesion molecules (ICAM-1, VCAM-1, E-selectin) and MHC class II are upregulated leading to recruitment of proinflammatory cells, and induction of immunoglobulins, and complement. Platelets become activated and “stickier,” causing small vessel occlusion, and containment of infections. In addition, it induces macrophages and endothelial cells to secrete chemokines and promotes apoptosis of target cells. TNFα has a potent paracrine function, inducing (via a NFκB-mediated mechanism) the secretion of pro-inflammatory cytokines such as IL-1, IL-6, and GM-CSF, and also stimulates the production of various chemokines, including RANTES, IL-8, MCP-1, and MIP-1α. Finally, TNFα plays a role in angiogenesis, which is critical to the growth and propagation of the rheumatoid synovium.

Immune-mediated inflammatory diseases (IMIDs) are triggered by an abnormal production of pro-inflammatory cytokines, including TNFα. These diseases include: rheumatoid arthritis (RA), inflammatory bowel disease (IBD) such as Crohn's disease, chronic plaque psoriasis, psoriatic arthritis, vasculitis, and ankylosing spondylitis.

New developments in the treatment of immune-mediated inflammatory diseases have arisen from basic research in cytokine expression and signaling that identified two key players in the pathophysiology of several diseases in this category: TNFα and interleukin interleukin-1 (IL-1). For example, both cytokines have been found to be elevated in the serum, synovium, and synovial fluid of patients with RA. Moreover, TNFα and IL-1 are capable of inducing and augmenting joint damage in experimental models of arthritis. Such findings led to the development of strategies to block/antagonize their effects, and specific targeting by biological agents has become possible in the recent years. This first generation of products includes monoclonal antibodies (mAb) and soluble receptor fusion proteins, all acting to compete with receptor for binding of the cytokine.

To date, the US FDA has approved three TNFα inhibitors/blocking agents for clinical use in the treatment of several IMIDs, including psoriasis and psoriatic arthritis. They are: Etanercept (Enbrel®, Amgen-Wyeth), a fully human chimeric protein of TNF receptor II fused to the Fc component of human IgG1, Infliximab (Remicade®, Centocor), a chimeric monoclonal antibody, and Adalimumab (Humira®, Abbott) a fully human IgG1 anti-TNFα monoclonal antibody. The proven initial efficacy and safety profile of these agents has to be weighed against the concerns for declining efficacy over time, the high cost of these biopharmaceutical agents and the limitations imposed by the current needle delivery for these agents.

An animal model developed by Boyman and colleagues (Boyman et al., J. Exp. Med. 199:731-736 [2004]) demonstrated the essential role for resident T-cells and TNFα in the spontaneous development of psoriasis. In this model, symptomless pre-psoriatic human skin lesions are grafted onto transgenic AGR129 mice. The group showed that blocking of T cells lead to inhibition of psoriasis development. It also showed that application of neutralizing anti-human TNFα monoclonal antibody (Infliximab, i.v.) or TNF receptor fusion protein (Etanercept, s.c.) led to dramatic inhibition of psoriatic phenotype development. These results have served to support the notion that TNFα antagonists have a direct effect in IMIDs disease development.

Unfortunately, more than one third of patients suffering from any of the approved IMIDs indications do not benefit clinically from anti-TNF treatment (Wong et al., Clin. Immunol. 126:121-136 [2008]). Efforts continue in the development of novel therapeutics to address this medical area. There are a number of “smart” TNFα antagonists currently in clinical trials, including: various anti-TNF monoclonal antibodies, pegylated antibody fragments or truncated TNF receptor molecules. In addition, a small-molecule inhibitor that promotes TNFα subunit disassembly of the trimeric cytokine has shown promising in vitro results (He et al., Science 310:1022-1025 [2005]) but no further clinical development has been reported.

Psoriasis is a common skin disorder that affects approximately 2.8 percent of the population (Linden and Weinstein Am. J. Med. 107:595-605 [1999]). An estimated 4.5 million people in the US suffer from psoriasis and 1.5 million have moderate to severe plaque psoriasis. The disease is characterized by chronic inflammation of the skin. This inflammation helps drive the formation of red, itchy skin plaques that are often painful and disfiguring. TNF-α plays a critical role in their formation and continued existence because it induces synthesis if IL-1 and IL-8, the triggers of inflammation. TNF-α concentrations are higher in psoriatic lesions than in unaffected skin of psoriatic patients and tend to decline with clearing of the lesions after effective therapy (Mussi et al., J. Biol. Regul. Homeost. Agents 11:115-118 [1997]). TNF-α also promotes keratinocyte proliferation and angiogenesis (Asadullah et al., Drugs today 35:913-924 [1999]), and thus inhibiting this cytokine should halt the disease at multiple stages.

Traditionally, first-line treatment of moderate psoriasis has consisted of topical agents because they are often less invasive than systemic therapy, with a low incidence of the most serious side effects, such as renal or hepatic failure (Kincaid (2005) Drug discovery today 10:884). Some agents such as: antithyroid thioureylenes, propylthiouracil and methimazole, are effective in the treatment of patients with psoriasis with a significant number of patients showing clearing or near clearing of their lesions after a several weeks of treatment. Systemic treatment with the new anti-TNFα biologics: Enbrel, Remicade, and Humira, has been approved for moderate to severe chronic plaque psoriasis. Patients treated with such agents very often show marked improvement in their disease with major clearing in several instances (Chaudhari et al., Lancet 357:1842-1847 [2001]; Leonardi et al., N. Engl. J. Med. 349:2014-2022 [2003]). But present day therapy of the disease is not particularly satisfactory and the many therapies currently in use are associated with significant cumulative toxicity (Gottlieb et al., J Am Acad Dermatol. 48:829-835 [2003]). Risks associated with TNFα blocking agents include serious infections, malignancies, anaphylaxis, hepatitis B reactivation, demyelinating disease, cytopenias, heart failure, and lupus-like syndrome. In addition, there is observed loss of clinical benefit after the drugs are stopped, and a small proportion of patients develop antibodies to the biological agents which is likely to limit their efficacy with repeated use.

Proteins such as the engineered modified variant BBPIs which are much smaller in size and thus presumed to be much less immunogenic should significantly reduce concerns about development of neutralizing antibodies in patients. BBPI molecules, because of their reduced molecular weight in comparison to antibodies or proteins fusions, will likely be more amenable to delivery via a topical route, or via less invasive systemic administrations like subcutaneous injections or needleless delivery methods.

The invention provides personal care compositions comprising a modified variant TNF-BBPI for use in skin and/or hair care. In some embodiments, the personal care composition comprising a TNF-BBPI is used for promoting hair growth and/or improving the condition of the skin of a subject suffering from a dermatological inflammatory disorder. In some embodiments, the inflammatory skin disorder is chosen from dermatitis, eczema, psoriasis, acne, rosacea and hives. In some embodiments, the dermatological inflammatory disorder is psoriasis. Thus, the invention provides for personal skin care and/or hair care compositions. The TNF-BBPIs comprised in the personal care compositions of the invention are modified variant BBPIs in which the equivalent chymotrypsin loop of the precursor scaffold of the TNF-5-BBPI has been replaced by a TNF variant peptide, and which further comprise at least one amino acid substitution as described in sections 5.4.1 and 5.4.2. In the present invention, binding of the modified variant TNF-BBPI to TNFα diverts TNFα from interacting with its cognate receptor and inhibits the TNFα-induced inflammation of the scalp and preventing hair loss. In some embodiments, binding of the modified variant TNF-BBPI to TNFα diminishes the inflammation of the skin and improves the condition of the skin in a subject suffering from a dermatological inflammatory disorder recited herein e.g. psoriasis. However, it is not intended that the present invention be limited to any particular mechanism.

In some embodiments, the TNF variant peptide comprised in the TNF-BBPI composition is chosen from RYWQDIP (T1; SEQ ID NO:474), APEPILA (T2; SEQ ID NO:475), DMIMVSI (T3; SEQ ID NO:476), WTPKPTQ (SEQ ID NO:583), ATFPNQS (SEQ ID NO:584), ASTVGGL (SEQ ID NO:585), TMLPYRP (SEQ ID NO:586), AWHSPSV (SEQ ID NO:587), TQSFSS (SEQ ID NO:588), THKNTLR (SEQ ID NO:589), GQTHFHV (SEQ ID NO:590), LPILTQT (SEQ ID NO:591), SILPVSH (SEQ ID NO:592), SQPIPI (SEQ ID NO:593), and QPLRKLP (SEQ ID NO:594). In other embodiments, the TNF variant peptide is chosen from SEQ ID NOS:483, 484 AND 485.

The scaffold in which the variant TNF peptide is introduced to replace the equivalent chymotrypsin loop is chosen from the scaffolds of the soybean inhibitor from Glycine max (BBI; SEQ ID NO:13) or the mature and truncated form thereof (SEQ ID NO:185), the inhibitor from Dolichos biflorus (BBdb; SEQ ID NO:449), the soybean inhibitor D-II from Glycine max (BBsb3; SEQ ID NO:450), the inhibitor from Torresea (Amburana) cearensis (BBtc; SEQ ID NO:451), the BBI-AV scaffold of (SEQ ID NO:186), the BBIt-AV scaffold of (SEQ ID NO:187), the BBdb-AV scaffold of (SEQ ID NO:452), the BBsb3-AV scaffold of (SEQ ID NO:453), the BBtc-AV scaffold of (SEQ ID NO:454), the BBIt-VEGK scaffold of (SEQ ID NO:640), the BBIt-VEGT scaffold of (SEQ ID NO:641) and the BBIt-VEGKD scaffold of (SE ID NO:642). In addition, any wild-type BBPI precursor scaffolds, such as those disclosed by Prakash et al. (supra), may be used to generate variant BBPI scaffolds. In some embodiments, the scaffold of the VEGF-BBPI is that of SEQ ID NO:187. In some embodiments, the backbone of the modified variant TNF-BBPI comprises at least one amino acid substitution at least at one amino acid position chosen from positions equivalent to 1, 4, 5, 11, 13, 18, 25, 27, 29, 31, 38, 40, 50, 52, 55, and 65 of the variant BBI of SEQ ID NO:187, as recited in section 5.4.1. In other embodiments, the backbone of the modified variant TNF-BBPI comprises a combination of amino acid substitutions chosen from a combination two, three, four, five, six, seven or eight amino acid substitutions as recited above in section 5.4.2.

In other embodiments, the backbone of the modified variant TNF-BBPI comprises a combination of amino acid substitutions chosen from a combination of amino acid substitutions chosen from 13I-29P-50T-52A, 13I-40K-50T-52A, 13I-25K-29P-52K, 13I-29P-40K-50T-52A, 13I-25R-27A-29P-31A-50K-52T, 13I-25R-27A-29P-31A-40H-50K-52T, 13I-25K-27A-29R-31E-40K-50Q-52Q, 13I-25K-27A-29R-31A-40H-50R-52L at equivalent positions in SEQ ID NO:187.

In some embodiments, the personal care compositions of the invention comprises a BBPI in which the equivalent chymotrypsin loop of the precursor scaffold is replaced with a TNF variant peptide chosen from SEQ ID NOS:474, 475 And 476,

wherein the scaffold is that of SEQ ID NO:187, and which comprises a combination of amino acid substitutions 13I-25R-27A-29P-31A-50K-52T.

In some embodiments, the personal care compositions comprise a TNF-BBPI chosen from the TNF-BBPIs of SEQ ID NOS:647, 648, and 649.

In some embodiments, the invention provides a personal care composition comprising a TNF-BBPI that binds to TNF. In alternative embodiments, the binding of the TNF-BBPI to TNF blocks the downstream activity of TNF. In some embodiments, the composition is capable of modulating inflammation.

In some embodiments, the personal care composition comprising a modified variant BBPI-TNF is for use in skin care. In some embodiments, the skin care compositions are for use in improving the appearance and/or condition of skin in a subject suffering from a skin disorder. Thus, in some embodiments, the personal care compositions for use in skin care include cosmetic compositions. In some embodiments, the skin disorder is an inflammatory skin disorder. In some embodiments, the inflammatory skin disorder is psoriasis.

In one embodiment, the personal care composition comprising a TNF-BBPI is a skin care composition is chosen from skin creams, lotions, sprays, emulsions, colloidal suspensions, foams, aerosols, liquids, gels, sera, and solids. In another embodiment, the personal care composition is a skin care composition selected from moisturizing body washes, body washes, antimicrobial cleansers, skin protective creams, body lotions, facial creams, moisturizing creams, facial cleansing emulsions, facial gels, facial sera, surfactant-based facial cleansers, facial exfoliating gels, anti-acne treatments, facial toners, exfoliating creams, facial masks, after shave balms, pre-shave balms, tanning compositions, skin lightening compositions, skin redness reduction compositions, sunscreens, depilatories, hair growth inhibitors, and radioprotectives. Radioprotectives are chosen from non-water-resistant sunscreens, very water-resistant sunscreens, and water-in-silicone sunscreens.

In one embodiment, the personal care composition comprising a TNF-BBPI is a skin care composition comprising topically applied over-the-counter compositions, anti-fungal treatments, anti-acne treatments, skin protectants, sunscreens, deodorants, and antiperspirants. In other embodiments, the skin care composition is capable of lightening skin tone, reducing redness, preventing skin tone darkening or preventing color development.

The present invention also provides personal care compositions that are cosmetic compositions. In some preferred embodiments, the cosmetic compositions are selected from mascaras, pressed powder formulations, and foundations. In some preferred embodiments, the makeup compositions comprise at least one pigment.

In some preferred embodiments, the makeup composition comprising at least one pigment is a mascara selected from non-waterproof mascaras, waterproof mascaras, volumizing mascaras, lengthening mascaras, curling mascaras, anhydrous waterproof mascaras, water-based mascaras, and eyelash or eyebrow treatments.

In yet additional embodiments, the makeup compositions are pressed powder formulations selected from loose powders, blushes, eye shadows, and bronzing powders. In still further embodiments, the makeup compositions are foundations selected from water-in-oil foundations, water-in-silicone foundations, oil-in-water foundations, anhydrous makeup sticks, and cream-to-powder foundations.

In some embodiments, the personal skin care compositions comprise a modified variant TNF-BBPI, as set forth herein, and a physiologically acceptable carrier or excipient. Preferably, the TNF-BBPI is present in an amount of about 0.0001% to about 5% by weight based on the total weight of the composition. Also preferably, the TNF-BBPI is present in an amount of about 0.001% to about 0.5% by weight based on the total weight of the composition. The composition may be in the form of an emulsified vehicle, such as a nutrient cream or lotion, a stabilized gel or dispersion system, a treatment serum, a liposomal delivery system, a topical pack or mask, a surfactant-based cleansing system such as a shampoo or body wash, an aerosolized or sprayed dispersion or emulsion, a hair or skin conditioner, styling aid, or a pigmented product such as makeup, as well as other suitable make-up and cosmetic preparations. In some embodiments, the carrier is preferably at least one selected from the group consisting of water, propylene glycol, ethanol, propanol, glycerol, butylene glycol and polyethylene glycol.

In other embodiments, the invention provides a personal care composition comprising a modified variant BBPIs that comprises a TNF variant peptide (TNF-BBPI) for use in hair care.

In some embodiments, the hair care compositions find use in improving the appearance and/or condition of scalp skin in patients suffering from psoriasis.

In one embodiment, the hair care composition is selected from the group consisting of shampoos, conditioners, hair styling compositions, hair colorants, permanent wave formulations, creams, gels, mousses, sprays, emulsions, colloidal suspensions, liquids, foams, and solids.

In some embodiments, the hair care composition further comprises a radioprotective. As described for the personal skin care compositions the radioprotective is a sunscreen chosen from non-water-resistant sunscreens, very water-resistant sunscreens, and water-in-silicone sunscreens. In other embodiments, the radioprotective is a sunscreen chosen from non-water-resistant sunscreens, very water-resistant sunscreens, and water-in-silicone sunscreens.

In some embodiments, the personal hair care compositions comprise a modified variant VEGF-BBPI, as set forth herein, and a physiologically acceptable carrier or excipient. Preferably, the VEGF-BBPI is present in an amount of about 0.0001% to about 5% by weight based on the total weight of the composition. Also preferably, the VEGF-BBPI is present in an amount of about 0.001% to about 0.5% by weight based on the total weight of the composition. The composition may be in the form of an emulsified vehicle, such as a nutrient cream or lotion, a stabilized gel or dispersion system, a treatment serum, a liposomal delivery system, a topical pack or mask, a surfactant-based cleansing system such as a shampoo or body wash, an aerosolized or sprayed dispersion or emulsion, a hair or skin conditioner, styling aid, or a pigmented product such as makeup, as well as other suitable make-up and cosmetic preparations. In some embodiments, the carrier is preferably at least one selected from the group consisting of water, propylene glycol, ethanol, propanol, glycerol, butylene glycol and polyethylene glycol.

Formulations

In additional preferred embodiments, the present invention provides cosmetic and/or pharmaceutical compositions comprising at least one modified variant BBPI, as set forth herein, and a physiologically acceptable carrier or excipient. Preferably, the compound is present in an amount of about 0.0001% to about 5% by weight, based on the total weight of the composition. Also preferably, the compound is present in an amount of about 0.001% to about 0.5% by weight based on the total weight of the composition. The composition may be in the form of an emulsified vehicle, such as a nutrient cream or lotion, a stabilized gel or dispersion system, a treatment serum, a liposomal delivery system, a topical pack or mask, a surfactant-based cleansing system such as a shampoo or body wash, an aerosolized or sprayed dispersion or emulsion, a hair or skin conditioner, styling aid, or a pigmented product such as makeup. Preferably, the carrier is at least compound selected from the group consisting of water, propylene glycol, ethanol, propanol, glycerol, butylene glycol and polyethylene glycol.

It is contemplated that the present invention will find use in numerous personal care compositions. It is not intended that the present invention be limited to any particular format or type of composition. The following description provides exemplary, not limiting compositions comprising the following invention.

Emulsions comprises one group of customary, commonly-used cosmetics. The term “emulsion” is generally used in reference to a heterogeneous system of two liquids which are immiscible or miscible only to a limited extent with one another, which are usually referred to as “phases.” One phase is typically in the form of droplets (i.e., the “dispersed,” “discontinuous” or “internal” phase), while the other liquid forms a continuous (i.e., “coherent” or “external”) phase. Less common forms of application include multiple emulsions (i.e. those in which the droplets of the dispersed [or discontinuous] phase, comprise for their part droplets of a further dispersed phase, such as water/oil/water [W/O/W] emulsions and oil/water/oil [O/W/O] emulsions).

If the oil phase is finely distributed in the water phase, then this is an oil-in-water emulsion (O/W emulsion; e.g. milk). The basic character of an O/W emulsion is determined by the water. These emulsions are generally less greasy on the skin, are rather matting, and absorb more rapidly into the skin than W/O (water-in-oil) emulsions.

Those of skill in the art are familiar with a large number of options of formulating stable W/O preparations for cosmetic and/or dermatological uses, including such formulations as creams and ointments, which are spreadable in the range from room temperature to skin temperature, as well as lotions and milks, which are more flowable in this temperature range.

The stability of emulsions is dependent on their viscosity, in particular on the viscosity of the external phase. An emulsion becomes unstable when the finely dispersed particles collect together to form relatively large aggregates, and the droplets which are in contact coalesce. This process is referred to as “coalescence.” The more viscous the external phase of the emulsion, the slower the process of coalescence. Emulsions of “liquid” (=flowable) consistency are used in various cosmetics (e.g., skin care lotions, cleansing lotions, face lotions, hand lotions, etc.). These compositions generally have a viscosity of from about 2000 mPa·s to about 10,000 mPa·s. The stability of flowable emulsions is deserving of particular attention since the considerably greater mobility of the particles promotes more rapid coalescence.

It is known that liquid emulsions typically presently in use generally comprise thickeners and are not stable toward relatively high electrolyte concentrations. This is manifested in phase separation of the compositions. However, in some embodiments, it is desirable to use certain electrolytes (e.g., water-soluble UV filters), in order to be able to utilize the other physical, chemical or physiological properties thereof. Although in many cases appropriate choice of the emulsifier system can provide remedies to a certain extent, other disadvantages then arise just as often.

For example, some disadvantages result due to the fact that emulsifiers, like ultimately any chemical substance, may trigger allergic reactions or reactions based on oversensitivity (i.e., hypersensitivity) of the user. The use of customary cosmetic emulsifiers is generally entirely without risk, although for some individuals, “hypoallergenic” compositions are necessary and/or preferred. Indeed, in some particularly sensitive individuals, certain dermatoses are triggered by exposure to certain emulsifiers and simultaneous exposure to sunlight. Thus, as known to those in the art, in some compositions, particular emulsifiers are less preferred and/or are avoided.

It is possible to prepare emulsifier-free preparations. For example, some preparations have an oily phase which contains dispersed water droplets (i.e., it is similar to a W/O emulsion). Such systems are sometimes called “hydrodispersions” or “oleodispersions,” depending upon which is the disperse phase and which is the continuous phase.

For cosmetic technology, it is, however, neither necessary nor possible to dispense with emulsifiers altogether, especially since there is a certain choice of particularly mild emulsifiers.

In some liposomal embodiments, the liposomes comprise water and one or more ingredients capable of forming lipid bilayer vesicles that can hold one or more functional or active ingredient(s). Non-limiting examples of ingredients capable of forming lipid bilayer vesicles include: phospholipids, hydrogenated phosphatidylcholine, lecithin, cholesterol and sphingolipids. Preferred liposomes include, without limitation: a) lipoid liposome 0003 (composed of water and lecithin and glycerin); b) lipoid liposome 0300 (composed of water and phosphatidylcholine); c) lipoid liposome 0111 (composed of water, Ginkgo biloba leaf extract, denatured alcohol, hydrogenated lecithin and cholesterol); d) anti-irritant liposomes (composed of water, cola acuminata seed extract, bisabolol and phospholipids); e) vitamin C and E liposomes (composed of water, phospholipids, tocopheryl acetate and ascorbyl palmitate); f) firming liposomes (composed of water, butylene glycol, pyrus malus (Apple) fruit extract, phospholipids, tocopheryl acetate and carbomer); and g) moisturizing liposomes (composed of water, sodium PCA, tocopheryl acetate, xanthan gum, arginine, lysine, glycine and proline).

In other embodiments, the personal care composition of the present invention further comprise at least one active ingredient in addition to the scaffolds provide herein. There are numerous active ingredients known to those of skill in the art that find use in the personal care compositions of the present invention. Indeed, it is contemplated that any suitable active ingredient or combination of suitable active ingredients will find use in the present invention (See e.g., McCutcheon's Functional Materials, North American and International Editions, published by MC Publishing Co. [2003]). For example, in some embodiments, the personal care compositions herein comprise a skin care active ingredient at a level from about 0.0001% to about 20%, by weight of the composition. In another embodiment, the personal care compositions comprise a skin care active ingredient from about 0.001% to about 0.5%, by weight of the composition. In yet another embodiment, the personal care compositions comprise a skin care active ingredient from about 0.01% to about 2%, by weight of the composition.

Non-limiting examples of functional or active ingredients that can be delivered via liposomes include: vitamins and their derivatives, antioxidants, proteins and peptides, keratolytic agents, bioflavinoids, terpenoids, phytochemicals, and extracts of plant, marine or fermented origin. In a preferred embodiment, the composition further comprises a skin care or hair care active. Active ingredients can include any of a wide variety of ingredients such as are known in the art. (See e.g., McCutcheon's Functional Materials, North American and International Editions, (2003), published by MC Publishing Co.). Preferably, such actives include but are not limited to antioxidants, such as tocopheryl and ascorbyl derivatives, bioflavinoids, terpenoids, synthetics and the like, vitamins and vitamin derivatives, hydroxyl- and polyhydroxy acids and their derivatives, such as AHAs and BHAs and their reaction products, peptides and polypeptides and their derivatives, such as glycopeptides and lipophilized peptides, heat shock proteins and cytokines, enzymes and enzymes inhibitors and their derivatives, such as proteases, MMP inhibitors, catalases, glucose oxydase and superoxide dismutase, amino acids and their derivatives, bacterial, fungal and yeast fermentation products and their derivatives, including mushrooms, algae and seaweed and their derivatives, phytosterols and plant and plant part extracts and their derivatives and phospholipids and their derivatives, anti-dandruff agents such as zinc pyrithione and delivery systems containing them, as provided herein and/or known in the art.

In some preferred embodiments, the skin care active is selected from the group consisting of a Vitamin B3 component, panthenol, Vitamin E, Vitamin E acetate, retinol, retinyl propionate, retinyl palmitate, retinoic acid, Vitamin C, theobromine, alpha-hydroxyacid, farnesol, phytrantriol, salicylic acid, palmityl peptapeptide-3 and mixtures thereof. In some preferred embodiments, the Vitamin B3 component is niacinamide. In some embodiments, the compositions provided herein comprise a skin care active at a level from about 0.0001% to about 20%, preferably from about 0.001% to about 0.5%, more preferably from about 0.01% to about 1%, by weight.

Exemplary derivatives of the foregoing vitamin B₃ compounds include nicotinic acid esters, including non-vasodilating esters of nicotinic acid, nicotinyl amino acids, nicotinyl alcohol esters of carboxylic acids, nicotinic acid N-oxide and niacinamide N-oxide. Suitable esters of nicotinic acid include nicotinic acid esters of C₁-C₂₂, preferably C₁-C₁₆, more preferably C₁-C₆ alcohols. In these embodiments, the alcohols are suitably straight-chain or branched chain, cyclic or acyclic, saturated or unsaturated (including aromatic), and substituted or unsubstituted. The esters are preferably non-vasodilating.

Non-vasodilating esters of nicotinic acid include tocopherol nicotinate and inositol hexanicotinate; tocopherol nicotinate are preferred. A more complete description of vitamin B₃ compounds is provided in WO 98/22085. Preferred vitamin B₃ compounds include niacinamide and tocopherol nicotinate.

In additional embodiments, the skin care active comprises at least one retinoid. The retinoid is preferably retinol, retinol esters (e.g., C₂-C₂₂ alkyl esters of retinol, including retinyl palmitate, retinyl acetate, retinyl proprionate), retinal, and/or retinoic acid (including all-trans retinoic acid and/or 13-cis-retinoic acid), more preferably retinoids other than retinoic acid. These compounds are well known in the art and are commercially available from a number of sources (e.g., Sigma and Boehringer Mannheim). Preferred retinoids include retinol, retinyl palmitate, retinyl acetate, retinyl proprionate, retinal, retinoic acid and combinations thereof. More preferred are retinol, retinoic propionate, retinoic acid and retinyl palmitate. In some embodiments, the retinoid is included as a substantially pure material, while in other embodiments, it is provided as an extract obtained by suitable physical and/or chemical isolation from natural (e.g., plant) sources. When a retinoid is included in the compositions herein, it preferably comprises from about 0.005% to about 2%, preferably from about 0.01% to about 1% retinoid. Retinol is preferably used in an amount of from about 0.01% to about 0.15%; retinol esters are preferably used in an amount of from about 0.01% to about 2% (e.g., about 1%).

In still further embodiments of the present invention, antioxidants are incorporated in the personal care compositions. It is contemplated that any suitable antioxidants will find use in the personal care compositions of the present invention. Suitable antioxidants include, but are not limited to amino acids (e.g., glycine, histidine, tyrosine, and tryptophan) and derivatives thereof, imidazoles (e.g. urocanic acid) and derivatives thereof, peptides (e.g., D,L-carnosine, D-carnosine, and L-carnosine) and derivatives thereof (e.g., anserine), carotenoids, carotenes (e.g., α-carotene, β-carotene, and γ-lycopene) and derivatives thereof, chlorogenic acid and derivatives thereof, aurothioglucose, propylthiouracil and other thiols (e.g., thioredoxin, glutathione, cysteine, cystine, cystamine and the glycosyl, N-acetyl, methyl, ethyl, propyl, amyl, butyl and lauryl, palmitoyl, oleyl, g-linoleyl, cholesteryl and glyceryl esters thereof) and salts thereof, dilauryl thiodipropionate, distearyl thiodipropionate, thiodipropionic acid and derivatives thereof (e.g., esters, ethers, peptides, lipids, nucleotides, nucleosides and salts), and sulfoximine compounds (e.g. buthionine sulfoximines, homocysteine sulfoximine, buthionine sulfones, penta-, hexa-, and heptathionine sulfoximine) in very small tolerated doses (e.g., typically pmol to mmol/kg), chelating agents (e.g., α-hydroxy fatty acids, palmitic acid, phytic acid, and lactoferrin), α-hydroxy acids (e.g. citric acid, lactic acid, and malic acid), humic acid, bile acid, bile extracts, bilirubin, biliverdin, EDTA, EGTA and derivatives thereof, unsaturated fatty acids and derivatives thereof (e.g., linolenic acids, linoleic acid, oleic acid), folic acid and derivatives thereof, furfurylidenesorbitol and derivatives thereof, ubiquinone and ubiquinol and derivatives thereof, vitamin C and derivatives thereof (e.g., sodium ascorbyl phosphate, ascorbyl palmitate, Mg ascorbyl phosphate, and ascorbyl acetate), tocopherols and derivatives (e.g., vitamin E acetate), coniferyl benzoate of benzoin resin, ferulic acid, furfurylideneglucitol, carnosine, butylhydroxytoluene, butylhydroxyanisole, nordihydroguaiacic acid, nordihydroguaiaretic acid, trihydroxybutyrophenone, uric acid and derivatives thereof, mannose and derivatives thereof, zinc and derivatives thereof (e.g., ZnO, ZnSO4), selenium and derivatives thereof (e.g., selenomethionine), stilbenes and derivatives thereof (e.g., stilbene oxide, trans-stilbene oxide) and the derivatives thereof (e.g., salts, esters, ethers, sugars, nucleotides, nucleosides, peptides and lipids) of said active ingredients which are suitable for the intended use of the particular embodiment(s) of the present invention.

In some embodiments, the concentration of one or more antioxidant in the compositions of the present invention is preferably from about 0.001 to about 30% by weight, particularly preferably from about 0.05 to about 20% by weight, and more preferably from about 1 to about 10% by weight, based on the total weight of the preparation. In additional embodiments, in which vitamin E and/or its derivatives are utilized as anti-oxidant(s), the preferred range is from about 0.001 to about 10% by weight, based on the total weight of the formulation. However, it is not intended that the present invention be limited to any specific antioxidant concentration(s), as various concentrations will find use in the various embodiments of the present invention.

In yet some additional embodiments, the active ingredient(s) is/are catechins, bile esters of catechins, and/or aqueous or organic extracts from plants or sections of plants which have a content of catechins or bile esters of catechins (e.g., the leaves of the Theaceae plant family, in particular of the species Camellia sinensis [green tea]). Their typical ingredients (e.g., polyphenols or catechins, caffeine, vitamins, sugars, minerals, aminoacids, lipids) find particular use in some embodiments of the present invention.

In some embodiments, catechins find use in the present invention. Catechins are a group of compounds which are regarded as hydrogenated flavones or anthocyanidines, and are derivatives of “catechin” (catechol, 3,3′,4′,5,7-flavanpentol, 2-(3,4-dihydroxyphenyl)chroman-3,5,7-triol). Epicatechin ((2R,3R)-3,3′,4′,5,7-flavanpentol) is also an active ingredient that finds use in some embodiments of the present invention.

In yet additional embodiments, plant extracts with a content of catechin, in particular extracts of green tea (e.g., extracts from leaves of the plants of the genus Camellia, in particular those used for tea, such as C. sinenis, C. assamica, C. taliensis. and C. irrawadiensis and hybrids of these species with other species, such as C. japonica) find use in some personal care compositions of the present invention.

In some further embodiments, preferred active ingredients include polyphenols and catechins from the group (−)-catechin, (+)-catechin, (−)-catechin gallate, (−)-gallocatechin gallate, (+)-epicatechin, (−)-epicatechin, (−)-epicatechin gallate, (−)-epigallocatechin, and (−)-epigallocatechin gallate.

In some additional embodiments of the compositions of the present invention flavone and its derivatives (also often collectively called “flavones”) find used. These compounds have the following basic structure (substitution positions are shown):

Some of the more important flavones which find use in some personal care compositions of the present invention are provided below. However, it is not intended that the present invention be limited to any particular flavone.

FLAVONES OH Substitution Positions 3 5 7 8 2′ 3′ 4′ 5′ (a) Flavone − − − − − − − − Flavonol + − − − − − − − Chrysin − + + − − − − − Galangin + + + − − − − − Apigenin − + + − − − + − Fisetin + − + − − + + − Luteolin − + + − − + + − Kaempferol + + + − − − + − Quercetin + + + − − + + − Morin + + + − + − + − Robinetin + − + − − + + + Gossypetin + + + + − + + − Myricetin + + + − − + + + (b) Flavone − − − − − − − − Flavonol + − − − − − − − Chrysin − + + − − − − − Galangin + + + − − − − − Apigenin − + + − − − + − Fisetin + − + − − + + − Luteolin − + + − − + + − Kaempferol + + + − − − + − Quercetin + + + − − + + − Morin + + + − + − + − Robinetin + − + − − + + + Gossypetin + + + + − + + − Myricetin + + + − − + + +

In nature, flavones are usually present in glycosylated form.

In some further embodiments, the personal care compositions of the present invention comprise at least one flavonoids having generic structural formula:

where Z₁ to Z₇, independently of one another, are chosen from the group consisting of H, OH, alkoxy and hydroxyalkoxy, where the alkoxy and hydroxyalkoxy groups can be branched or unbranched and have 1 to 18 carbon atoms, and where Gly is chosen from the group of mono- and oligoglycoside radicals.

In some alternative embodiments, the personal care compositions of the present invention comprise at least one flavonoids having the generic structural formula:

where Z₁ to Z₆, independently of one another, are chosen from the group consisting of H, OH, alkoxy and hydroxyalkoxy, where the alkoxy and hydroxyalkoxy groups may be branched or unbranched and have 1 to 18 carbon atoms, where Gly is chosen from the group mono and oligoglycoside radicals.

In some preferred embodiments, the composition has the generic structural formula

where Gly₁, Gly₂ and Gly₃, independently of one another, are monoglycoside radicals. Gly₂ and Gly₃ may also, individually or together, represent saturations by hydrogen atoms. In some preferred embodiments, Gly₁, Gly₂ and Gly₃, independently of one another, are selected from the group of hexosyl radicals, in particular the rhamnosyl radicals and glucosyl radicals. However, hexosyl radicals, for example allosyl, altrosyl, galactosyl, gulosyl, idosyl, mannosyl and talosyl, also find use in some embodiments of the present invention. In yet additional embodiments, pentosyl radicals find use in some personal care compositions of the present invention.

In some embodiments, Z₁ to Z₅ are, independently of one another, advantageously chosen from the group consisting of H, OH, methoxy, ethoxy and 2-hydroxyethoxy, and the flavone glycosides have the structure:

In some embodiments, the flavone glycosides provided in some of the personal care compositions of the present invention have the following structure:

where Gly₁, Gly₂ and Gly₃, independently of one another, are monoglycoside radicals. Gly₂ and Gly₃ can also, individually or together, represent saturations by hydrogen atoms. In alternative embodiments, Gly₁, Gly₂ and Gly₃, independently of one another, are selected from the group of hexosyl radicals, in particular of rhamnosyl radicals and glucosyl radicals. However, other hexosyl radicals, for example allosyl, altrosyl, galactosyl, gulosyl, idosyl, mannosyl and talosyl, find use in some embodiments of the present invention. In addition, in some embodiments, pentosyl radicals find use in the present invention. In some preferred embodiments, the personal care compositions of the present invention comprise one or more flavone glucoside selected from the group consisting of a-glucosylrutin, a-glucosylmyricetin, a-glucosylisoquercitrin, a-glucosylisoquercetin and a-glucosylquercitrin. In some particularly preferred embodiments, the flavone glucoside is a-glucosylrutin.

In yet some additional embodiments, the personal care compositions of the present invention comprise at least one naringin (e.g., aurantin, naringenin-7-rhamno-glucoside), hesperidin 3′,5,7-trihydroxy-4′-methoxyflavanone-7-rutinoside, hesperidoside, hesperetin-7-O-rutinoside), rutin (3,3′,4′,5,7-pentahydroxyflavone-3-rutinoside, quercetin-3-rutinoside, sophorin, birutan, rutabion, taurutin, phytomelin, melin), troxerutin (3,5-dihydroxy-3′,4′,7-tris(2-hydroxyethoxy)flavone-3-(6-O-(6-deoxy-a-L-mannopyranosyl)-b-D-glucopyranoside)), monoxerutin (3,3′,4′,5-tetrahydroxy-7-(2-hydroxyethoxy)flavone-3-(6-O-(6-deoxy-a-L-mannopyranosyl)-b-D-g lucopyranoside)), dihydrorobinetin (3,3′,4′,5′,7-pentahydroxyflavanone), taxifolin (3,3′,4′,5,7-pentahydroxyflavanone), eriodictyol-7-g lucoside (3′,4′,5,7-tetrahydroxyflavanone-7 glucoside), flavanomarein (3′,4′,7,8-tetrahydroxyflavanone-7 glucoside), and/or isoquercetin (3,3′,4′,5,7-pentahydroxyflavanone-3-(b-D-glucopyranoside). In some yet further embodiments, the active ingredient is selected from the group consisting of ubiquinones and plastoquinones. Ubiquinones are characterized by the structural formula:

Ubiquinones are the most widespread and the most investigated bioquinones. Ubiquinones are referred to, depending on the number of isoprene units linked in the side chain, as Q-1, Q-2, Q-3 etc., or according to the number of carbon atoms, as U-5, U-10, U-15 etc. They preferably arise with certain chain lengths (e.g. in some microorganisms and yeasts where n=6). In most mammals, including humans, Q10 predominates. Coenzyme Q10 finds particular use in some embodiments of the present invention. Its structural formula is:

Plastoquinones have the general structural formula:

Plastoquinones differ in the number n of isoprene radicals and are referred to accordingly (e.g. PQ-9 [n=9]). In addition, other plastoquinones with varying substituents on the quinone ring exist in some embodiments.

In yet additional embodiments, the present invention provides preparations suitable for use as deodorants and/or antiperspirants. It is contemplated that any of the active ingredients which commonly find use in such preparations will also find use in various embodiments of the present invention. Additional components that are commonly used in such preparations also find use in various embodiments of the present invention. Examples of such actives and inactive compounds include, but are not limited to odor maskers (e.g., perfumes), odor absorber (e.g., phyllosilicates described in DE-P 40 09 347); as well as montmorillonite, kaolinite, illite, beidellite, nontronite, saponite, hectorite, bentonite, smectite, and zinc salts of ricinoleic acid. In some embodiments of the present invention, the range of active ingredients (i.e., one or more compounds) in such preparations is preferably from about 0.001 to about 30% by weight; more preferably from about 0.05 to about 20% by weight; and most particularly in the range of from about 1 to about 10% by weight, based on the total weight of the preparation.

In some embodiments, the compositions of the present invention comprise safe and effective amounts of a dermatologically acceptable carrier that is suitable for topical application to the skin or hair within which the essential materials and optional other materials are incorporated to enable the essential materials and optional components to be delivered to the skin or hair at an appropriate concentration. Thus, in some embodiments, the carrier acts as a diluent, dispersant, solvent or the like for the essential components, ensuring that these components can be applied and distributed evenly over the selected target at an appropriate concentration.

In further embodiments, an effective amount of one or more compounds described herein is also be included in compositions to be applied to keratinous materials such as nails and hair, including but not limited to those useful as hair spray compositions, hair styling compositions, hair shampooing and/or conditioning compositions, compositions applied for the purpose of hair growth regulation and compositions applied to the hair and scalp for the purpose of treating seborrhoea, dermatitis and/or dandruff.

In yet additional embodiments, an effective amount of one or more compounds described herein is included in compositions suitable for topical application to the skin or hair. These compositions are provided in any suitable form, including but not limited to creams, lotions, gels, suspensions dispersions, microemulsions, nanodispersions, microspheres, hydrogels, emulsions (e.g., oil-in-water and water-in-oil, as well as multiple emulsions), and multilaminar gels and the like (See e.g., Schlossman et al., The Chemistry and Manufacture of Cosmetics, [1998], incorporated by reference, herein). In some embodiments, the compositions are formulated as aqueous or silicone compositions, while in other embodiments they are formulated as emulsions of one or more oil phases in an aqueous continuous phase (or an aqueous phase in an oil phase).

The type of carrier utilized in the present invention depends on the type of product form desired for the composition. The carrier can be solid, semi-solid or liquid. Suitable carriers include liquids, semi-solids (e.g., creams, lotions, gels, sticks, ointments, and pastes), sprays and mousses. Preferably the carrier is in the form of a lotion, cream or a gel, more preferably one which has a sufficient thickness or yield point to prevent the particles from sedimenting. In some embodiments, the carrier is inert, while in other embodiments it provides dermatological benefits. In some embodiments, the carrier is applied directly to the skin and/or hair, while in other embodiments, it is applied via a woven or non-woven wipe or cloth. In yet other embodiments, it is in the form of a patch, mask or wrap. In still further embodiments, it is aerosolized or otherwise sprayed or pumped onto the skin and/or hair. The carrier chosen is physically and chemically compatible with the essential components described herein, and should not unduly impair stability, efficacy or other use benefits associated with the compositions of the present invention.

Preferred carriers contain a dermatologically acceptable, hydrophilic diluent. Suitable hydrophilic diluents include water, organic hydrophilic diluents such as C₂-C₁₀, preferably C₂-C₆, preferably, C₃-C₆ monohydric alcohols and low molecular weight glycols and polyols, including propylene glycol, polyethylene glycol polypropylene glycol, glycerol, butylene glycol, 1,2,4-butanetriol, sorbitol esters, 1,2,6-hexametriol, pentylene glycol, hexylene glycol, sorbitol esters, ethoxylated ethers, propoxylated ethers, and combinations thereof. The diluent is preferably liquid. Water is a preferred diluent. The composition preferably comprises at least about 20% of the hydrophilic diluent.

In some embodiments, suitable carriers also comprise an emulsion comprising a hydrophilic phase, especially an aqueous phase, and a hydrophobic phase (e.g., a lipid, oil or oily material). As well known to those skilled in the art, the hydrophilic phase is dispersed in the hydrophobic phase, or vice versa, to form respectively hydrophilic or hydrophobic dispersed and continuous phases, depending on the composition of ingredients. The term “dispersed phase” is a term well-known to one skilled in the art of emulsion technology, used in reference to the phase which exists as small particles or droplets that are suspended in and surrounded by a continuous phase. The dispersed phase is also known as the internal or discontinuous phase. The emulsion may be or comprise (e.g., in a triple or other multi-phase emulsion) an oil-in-water emulsion or a water-in-oil emulsion such as a water-in-silicone emulsion. Oil-in-water emulsions typically comprise from about 1% to about 60% (preferably about 1% to about 30%) of the dispersed hydrophobic phase and from about 1% to about 99% (preferably from about 10% to about 90%) of the continuous hydrophilic phase, while water-in-oil emulsions typically comprise from about 1% to about 98% (preferably from about 40% to about 90%) of the dispersed hydrophilic phase and from about 1% to about 50% (preferably about 1% to about 30%) of the continuous hydrophobic phase.

In further embodiments, the carrier also includes one or more components that facilitate penetration through the upper stratum corneum barrier to the lower levels of the skin. Examples of penetration enhancers include, but are not limited to, propylene glycol, azone, ethoxydiglycol, dimethyl isosorbide, urea, ethanol and dimethyl sulfoxide, as well as microemulsions, liposomes and nanoemulsions.

In some additional embodiments, the compositions of the present invention comprise humectants which are preferably present at a level of from about 0.01% to about 20%, preferably from about 0.1% to about 15% and preferably from about 0.5% to about 10%. Preferred humectants include, but are not limited to, compounds selected from polyhydric alcohols, sorbitol, glycerol, urea, betaine, D-panthenol, DL-panthenol, calcium pantothenate, royal jelly, panthetine, pantotheine, panthenyl ethyl ether, pangamic acid, pyridoxin, pantoyl lactose Vitamin B complex, sodium pyrrolidone carboxylic acid, hexane-1,2,6,-triol, guanidine or its derivatives, and mixtures thereof.

Suitable polyhydric alcohols for use herein include, but are not limited to polyalkylene glycols and preferably alkylene polyols and their derivatives, including propylene glycol, dipropylene glycol, polypropylene glycol, polyethylene glycol and derivatives thereof, sorbitol, hydroxypropyl sorbitol, erythritol, threitol, pentaerythritol, xylitol, glucitol, mannitol, pentylene glycol, hexylene glycol, butylene glycol (e.g., 1,3-butylene glycol), hexane triol (e.g., 1,2,6-hexanetriol), trimethylol propane, neopentyl glycol, glycerine, ethoxylated glycerine, propane-1,3 diol, propoxylated glycerine and mixtures thereof. The alkoxylated derivatives of any of the above polyhydric alcohols are also suitable for use herein. Preferred polyhydric alcohols of the present invention are selected from glycerine, butylene glycol, propylene glycol, pentylene glycol, hexylene glycol, dipropylene glycol, polyethylene glycol, hexane triol, ethoxylated glycerine and propoxylated glycerine and mixtures thereof.

Suitable humectants useful herein are sodium 2-pyrrolidone-5-carboxylate (NaPCA), guanidine; glycolic acid and glycolate salts (e.g., ammonium and quaternary alkyl ammonium); lactic acid and lactate salts (e.g., ammonium and quaternary alkyl ammonium); aloe vera in any of its variety of forms (e.g., aloe vera gel); hyaluronic acid and derivatives thereof (e.g., salt derivatives such as sodium hyaluronate); lactamide monoethanolamine; acetamide monoethanolamine; urea; betaine, panthenol and derivatives thereof; and mixtures thereof.

In some embodiments, at least part (up to about 5% by weight of composition) of a humectant is incorporated into the compositions of the present invention in the form of an admixture with a particulate cross-linked hydrophobic acrylate or methacrylate copolymer, itself preferably present in an amount of from about 0.1% to about 10%, which can be added either to the aqueous or disperse phase. This copolymer is particularly valuable for reducing shine and controlling oil while helping to provide effective moisturization benefits and is described in further detail in WO96/03964, incorporated herein by reference.

In some embodiments, the oil-in-water and water-in-oil compositions of the present invention comprise from about 0.05% to about 20%, preferably from about 1% to about 15%, preferably from about 2% to about 10%, preferably from about 2% to about 5% of a dermatologically acceptable emollient. Emollients tend to lubricate the skin, increase the smoothness and suppleness of the skin, prevent or relieve dryness of the skin and/or protect the skin. Emollients are typically water-immiscible, oily or waxy materials and emollients can confer aesthetic properties to a topical composition. A wide variety of suitable emollients are known (See e.g., Sagarin, Cosmetics, Science and Technology, 2nd Edition, Vol. 1, pp. 32-43 [1972]; and WO 00/24372), and find use herein, contains numerous examples of materials suitable as emollients. Additional emollients include, but are not limited to the following:

i) Straight and branched chain hydrocarbons having from about 7 to about 40 carbon atoms, such as mineral oils, dodecane, squalane, cholesterol, hydrogenated polyisobutylene, isohexadecane, isoeicosane, isooctahexacontane, isohexapentacontahectane, and the C₇-C₄₀ isoparaffins, which are C₇-C₄₀ branched hydrocarbons. Suitable branched chain hydrocarbons for use herein are selected from isopentacontaoctactane, petrolatum and mixtures thereof;

ii) C₁-C₃₀ fatty acid esters of C₁-C₃₀ carboxylic acids, C₁₂₋₁₅ alkyl benzoates and of C₂-C₃₀ dicarboxylic acids, e.g. isononyl isononanoate, isostearyl neopentanoate, isodecyl octanoate, isodecyl isononanoate, tridecyl isononanoate, myristyl octanoate, octyl pelargonate, octyl isononanoate, myristyl myristate, myristyl neopentanoate, myristyl octanoate, isopropyl myristate, myristyl propionate, isopropyl stearate, isopropyl isostearate, methyl isostearate, behenyl behenate, dioctyl maleate, diisopropyl adipate, and diisopropyl dilinoleate and mixtures thereof also find use in the present invention;

iii) C₁-C₃₀ mono- and poly-esters of sugars and related materials. These esters are derived from a sugar or polyol moiety and one or more carboxylic acid moieties. Depending on the constituent acid and sugar, these esters can be in either liquid or solid form at room temperature. Examples include: glucose tetraoleate, the galactose tetraesters of oleic acid, the sorbitol tetraoleate, sucrose tetraoleate, sucrose pentaoleate, sucrose hexaoleate, sucrose heptaoleate, sucrose octaoleate, sorbitol hexaester. Other materials include cottonseed oil or soybean oil fatty acid esters of sucrose. Other examples of such materials are described in WO 96/16636, incorporated by reference herein;

iv) Vegetable oils and hydrogenated vegetable oils. Examples of vegetable oils and hydrogenated vegetable oils include safflower oil, grapeseed oil, coconut oil, cottonseed oil, menhaden oil, palm kernel oil, palm oil, peanut oil, soybean oil, rapeseed oil, linseed oil, rice bran oil, pine oil, nut oil, sesame oil, sunflower seed oil, partially and fully hydrogenated oils from the foregoing sources and mixtures thereof;

v) Soluble or colloidally-soluble moisturizing agents. Examples include hyaluronic acid and chondroitin sulfate.

The term “lipid” is often used as a generic term to refer to fats, oils, waxes and the like. In addition, the terms “oil phase” and “lipid phase” are also used synonymously. However, oils and fats differ from one another in their polarity, which is difficult to define. It has been proposed to adopt the interfacial tension toward water as a measure of the polarity index of an oil or of an oily phase. Thus, it is contemplated that the interfacial tension be regarded as a suitable measure of the polarity of a given oil component. The “interfacial tension” is the force which acts on an imaginary line one meter in length in the interface between two phases. In this measurement, the lower the interfacial tension between the oily phase and water, the greater the polarity of the oily phase being analyzed. The physical unit for this interfacial tension is conventionally calculated from the force/length relationship and is usually expressed in mN/m (millinewtons divided by meters). It has a positive sign if it endeavours to reduce the interface. In the converse case, it has a negative sign. As used herein, lipids are regarded as “polar,” if their interfacial tension toward water is less than 30 mN/m.

“Polar oils” include those from the group of lecithins and of fatty acid triglycerides, namely the triglycerol esters of saturated and/or unsaturated, branched and/or unbranched alkane carboxylic acids having a chain length of from 8 to 24, in particular 12 to 18, carbon atoms. In some embodiments, the fatty acid triglycerides are chosen from the group consisting of synthetic, semi-synthetic and natural oils (e.g., olive oil, sunflower oil, soya oil, groundnut oil, rapeseed oil, almond oil, palm oil, coconut oil, castor oil, wheatgerm oil, grapeseed oil, thistle oil, evening primrose oil, macadamia nut oil and the like). However, is it not intended that the present invention be limited to compositions that contain particular polar oils. Additional examples of polar oils that find use in the present invention include the group of esters of saturated and/or unsaturated, branched and/or unbranched alkane carboxylic acids having a chain length of from 3 to 30 carbon atoms and saturated and/or unsaturated, branched and/or unbranched alcohols having a chain length of from 3 to 30 carbon atoms, and from the group of esters of aromatic carboxylic acids and saturated and/or unsaturated, branched and/or unbranched alcohols having a chain length of from 3 to 30 carbon atoms. In some embodiments, such ester oils are chosen from the group consisting of isopropyl myristate, isopropyl palmitate, isopropyl stearate, isopropyl oleate, n-butyl stearate, n-hexyl laurate, n-decyl oleate, isooctyl stearate, isononyl stearate, isononyl isononanoate, 2-ethylhexyl palmitate, 2-ethylhexyl laurate, 2-hexyldecyl stearate, 2-octyldodecyl palmitate, oleyl oleate, oleyl erucate, erucyl oleate, erucyl erucate and synthetic, semi-synthetic and natural mixtures of such esters (e.g., jojoba oil).

In addition, in some embodiments, the oily phase is chosen from the group consisting of dialkyl ethers, as well as saturated or unsaturated, and branched or unbranched alcohols. In some particularly preferred embodiments, the oily phase of the compositions of the preferred embodiments also contains C₁₂₋₁₅-alkyl benzoate, while in alternative embodiments, the preferred embodiments contains only the latter. In yet additional embodiments, the oil phase is chosen from the group of Guerbet alcohols (i.e., the group of alcohols named after Marcel Guerbet who first described their preparation). These alcohols are formed according to the equation:

by oxidation of an alcohol to an aldehyde, by aldol condensation of the aldehyde, elimination of water from the aldol and hydrogenation of the allyl aldehyde. Guerbet alcohols are liquid even at low temperatures and result in virtually no skin irritations. Thus, they find use as fatting, superfatting and also refatting constituents in skincare and hair care compositions. Indeed, the use of Guerbet alcohols is known in the cosmetic art. In these applications, the species are generally characterized as having the following structure:

In this structure, R₁ and R₂ are usually unbranched alkyl radicals. In some preferred embodiments of the present invention the following Guerbet alcohols in which R₁ is propyl, butyl, pentyl, hexyl, heptyl or octyl and/or R₂ is hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl or tetradecyl find use in the present invention. In additional embodiments, preferred Guerbet alcohols include 2-butyloctanol with the following chemical structure:

which is commercially available, for example, under the trade name ISOFOL® 12 (Condea Chemie GmbH), and 2-hexyldecanol with the following chemical structure:

which is commercially available, for example, under the trade name ISOFOL® 16 (Condea Chemie GmbH).

In additional embodiments, mixtures of Guerbet alcohols find use in compositions of the present invention. For example, mixtures of 2-butyloctanol and 2-hexyldecanol find use in some embodiments. The total amount of Guerbet alcohols in the finished cosmetic or dermatological preparations is selected from the of range up to about 25.0% by weight, preferably about 0.5 to about 15.0% by weight, based on the total weight of the preparations. However, it is not intended that the present invention be limited to any particular concentration nor range of concentrations, as those of skill in the art know how to prepare compositions having suitable concentrations for the desired compositions and their use(s). In addition, it is contemplated that any mixtures of oil and/or wax components will find use in the present invention. For example, in some embodiments, waxes (e.g., cetyl palmitate) find use as the sole lipid component of the oil phase. In additional embodiments, nonpolar oils (e.g., those which are chosen from the group of branched and unbranched hydrocarbons and hydrocarbon waxes, in particular VASELINE® [i.e., petrolatum], paraffin oil, squalane and squalene, polyolefins and hydrogenated polyisobutenes find use in the present invention. In some embodiments containing polyolefins, polydecenes are the preferred substances.

Fatty and/or wax components which find use in embodiments of the present invention include but are not limited to vegetable waxes, animal waxes, mineral waxes and petrochemical waxes. Examples which particularly preferred waxes include candelilla wax, carnauba wax, japan wax, esparto grass wax, cork wax, guaruma wax, rice germ oil wax, sugar cane wax, berry wax, ouricury wax, montan wax, jojoba wax, shea butter, beeswax, shellac wax, spermaceti, lanolin (wool wax), uropygial grease, ceresin, ozokerite (earth wax), paraffin waxes and microcrystalline waxes.

Additional fatty and/or wax components that find use in the present invention include chemically modified waxes and/or synthetic waxes (e.g., those commercially available under the trade names SYNCROWAX® HRC [glyceryl tribehenate] and SYNSCROWAX® AW 1C [C₁₈-C₃₆ fatty acid], which are available from CRODA GmbH), and montan ester waxes, Sasol waxes, hydrogenated jojoba waxes, synthetic or modified beeswaxes (e.g., dimethicone copolyol beeswax and/or C₃₀₋₅₀ alkyl beeswax), polyalkylene waxes, polyethylene glycol waxes, as well as chemically modified fats (e.g., hydrogenated vegetable oils, such as hydrogenated castor oil and/or hydrogenated coconut fatty glycerides), triglycerides (e.g., trihydroxystearin, fatty acids, fatty acid esters, and glycol esters, such as, C₂₀-C₄₀-alkyl stearate, C₂₀-C₄₀-alkylhydroxystearoyl stearate and/or glycol montanate). In further embodiments, the present invention comprises certain organosilicone compounds, which have similar physical properties to the specified fatty and/or wax components (e.g., stearoxytrimethylsilane). In additional embodiments, the fatty and/or wax components are provided individually, while in still further embodiments, they are provided as a mixture. Indeed, it is intended that any desired mixture of such oil and/or wax components will find use in various embodiments of the present invention.

In some embodiments, the oily phase is selected from the group consisting of 2-ethylhexyl isostearate, octyldodecanol, isotridecyl isononanoate, isoeicosane, 2-ethylhexyl cocoate, C₁₂-C₁₅-alkyl benzoate, caprylic/capric triglyceride, and dicaprylyl ether. In alternative embodiments, mixtures of various oily phases are provided, including but not limited to mixtures comprising one or more of octyldodecanol, caprylic/capric triglyceride, dicaprylyl ether, C₁₂-C₁₅-alkyl benzoate, 2-ethylhexyl isostearate, isotridecyl isononanoate. The following table provides a list of lipids which find use alone or in combination with other lipids in various embodiments of the present invention. The corresponding interfacial tensions toward water are given in the last column. However, it is not intended that the present invention be limited to these specific components, as other components find use in various embodiments of the present invention, including mixtures of greater or lesser polar components and the like.

LIPIDS Trade name Article II. INCI name (m/Nm) ISOFOL ® 14 T Butyl Decanol + Hexyl Decanol + 27.6 Hexyl Octanol + Butyl Octanol ISOFOL ® 16 Hexyl Decanol 24.3 EUTANOL ® G Octyldodecanol 24.8 CETIOL ® OE Dicaprylyl Ether 22.1 MIGLYOL ® 812 Caprylic/Capric Triglyceride 21.3 CEGESOFT ® C24 Octyl Palmitate 23.1 Isopropyl stearate Isopropyl Stearate 21.9 ESTOL ® 1540 EHC Octyl Octanoate 30.0 FINSOLV ® TN C₁₂—C₁₅ Alkyl Benzoate 21.8 CETIOL ® SN Cetearyl Isonoanoate 28.6 DERMOFEEL ® BGC Butylene Glycol Dicaprylate/Dicapate 21.5 TRIVENT ® OCG Tricaprylin 20.2 MOD Octyldodeceyl Myristate 22.1 COSMACOL ® ETI Di-C₁₂—C₁₃ Alkyl Tartrate 29.4 MIGLYCOL ® 829 Caprylic/Capric Diglyceryl Succinate 29.5 PRISORINE ® 2036 Octyl Isostearate 29.7 TEGOSOFT ® SH Stearyl Heptanoate 28.7 ABIL ® Wax 9840 Cetyl Dimethicone 25.1 CETIOL ® LC Coco-Caprylate/Caprate 24.8 IPP Isopropyl Palmitate 22.5 LUVITOL ® EHO Cetearyl Octanoate 28.6 CETIOL ® 868 Octyl Stearate 28.4

In some embodiments, some or all of the oil phase of the preparations are selected from the group consisting of cyclic and/or linear silicones which are also often referred to as “silicone oils.” In some embodiments, these silicones or silicone oils are present as monomers which are generally characterized by structural elements as follows:

Silicones having two or more siloxyl units which find use in some embodiments of the present invention are generally characterized by structural elements as follows:

where the silicon atoms may be substituted by identical or different alkyl radicals and/or aryl radicals, which are represented in general terms by the radicals R₁ to R₄, where the number of different radicals is not necessarily limited to 4 and may assume values from 2 to 200,000.

Cyclic silicones to be used advantageously according to the invention are generally characterized by the structural elements as follows:

where the silicon atoms may be substituted by identical or different alkyl radicals and/or aryl radicals, which are represented here in general terms by the radicals R₁ to R₄, where the number of different radicals is not necessarily limited to 4. n can assume values of 3/2 to 20. Fractional values for “n” take into consideration that uneven numbers of siloxyl groups may be present in the cycle.

In some embodiments, phenyltrimethicone is selected as silicone oil. Other silicone oils suitable for use in various embodiments of the present invention include, but are not limited to dimethicone, phenyldimethicone, cyclomethicone (octamethylcyclotetrasiloxane), hexamethylcyclotrisiloxane, polydimethylsiloxane, poly(methylphenylsiloxane), cetyldimethicone, and behenoxydimethicone. In alternative embodiments, mixtures of these compounds find use in the present invention, including but not limited to mixtures of cyclomethicone and isotridecyl isononanoate, and mixtures of cyclomethicone and 2-ethylhexyl isostearate. It yet additional embodiments, silicone oils of similar constitution, such as the compounds referred to above whose organic side chains have been derivatized (e.g., polyethoxylated and/or polypropoxylated) find use in the present invention. These include, but are not limited to such compounds as polysiloxane-polyalkyl-polyether copolymers such as cetyldimethicone copolyol (i.e., cetyldimethicone copolyol (and) polyglyceryl-4 isostearate (and) hexyl laurate). Indeed, it is not intended that the present invention be limited to any specific silicone oil nor mixture of silicone oils, as various oils find use in various embodiments of the present invention.

In additional embodiments, water in oil (W/O) emulsions find use in the present invention. In some embodiments, W/O emulsifiers are used with or without additional co-emulsifiers. In still further embodiments, W/O emulsions of the present further comprise one or more emulsifiers, including, but not limited to one or more of the following compounds: lecithin, lanolin, microcrystalline wax (Cera microcristallina) in a mixture with paraffin oil (Paraffinum liquidum), ozokerite, hydrogenated castor oil, polyglyceryl-3 oleate, wool wax acid mixtures, wool wax alcohol mixtures, pentaerythrithyl isostearate, polyglyceryl-3 diisostearate, beeswax (Cera alba) and stearic acid, sodium dihydroxycetylphosphate in a mixture with isopropyl hydroxycetyl ether, methylglucose dioleate, methylglucose dioleate in a mixture with hydroxystearate and beeswax, mineral oil in a mixture with petrolatum and ozokerite and glyceryl oleate and lanolin alcohol, petrolatum in a mixture with ozokerite and hydrogenated castor oil and glyceryl isostearate and polyglyceyl-3 oleate, PEG-7 hydrogenated castor oil, ozokerite and hydrogenated castor oil, polyglyceryl-4 isostearate, polyglyceryl-4 isostearate in a mixture with cetyldimethicone copolyol and hexyl laurate, laurylmethicone copolyol, cetyldimethicone copolyol, acrylate/C₁₀-C₃₀-alkyl acrylate crosspolymer, Poloxamer 101, polyglyceryl-2 dipolyhydroxystearate, polyglyceryl-3 diisostearate, polyglyceryl-4 dipolyhydroxystearate, PEG-30 dipolyhydroxystearate, diisostearoyl polyglyceryl-3 diisostearate, polyglyceryl-2 dipolyhydroxystearate, polyglyceryl-3 dipolyhydroxystearate, polyglyceryl-4 dipolyhydroxystearate, polyglyceryl-3 dioleate.

In yet additional embodiments of the present invention, W/O emulsions of the present invention comprise one or more coemulsifiers, including, but not limited to the following:

glyceryl stearate in a mixture with ceteareth-20, ceteareth-25, ceteareth-6 in a mixture with stearyl alcohol, cetylstearyl alcohol in a mixture with PEG-40 castor oil and sodium cetylstearyl sulfate, triceteareth-4 phosphate, sodium cetylstearyl sulfate, lecithin trilaureth-4 phosphate, laureth-4 phosphate, stearic acid, propylene glycol stearate SE, PEG-25 hydrogenated castor oil, PEG-54 hydrogenated castor oil, PEG-6 caprylic/capric glycerides, glyceryl oleate in a mixture with propylene glycol, ceteth-2, ceteth-20, polysorbate 60, glyceryl stearate in a mixture with PEG-100 stearate, laureth-4, ceteareth-3, isostearyl glyceryl ether, cetylstearyl alcohol in a mixture with sodium cetylstearyl sulfate, laureth-23, steareth-2, glyceryl stearate in a mixture with PEG-30 stearate, PEG-40 stearate, glycol distearate, PEG-22 dodecyl glycol copolymer, polyglyceryl-2 PEG-4 stearate, ceteareth-20, methylglucose sesquistearate, steareth-10, PEG-20 stearate, steareth-2 in a mixture with PEG-8 distearate, steareth-21, steareth-20, isosteareth-20, PEG-45/dodecyl glycol copolymer, methoxy-PEG-22/dodecyl glycol copolymer, PEG-20 glyceryl stearate, PEG-8 beeswax, polyglyceryl-2 laurate, isostearyl diglyceryl succinate, stearamidopropyl PG dimonium chloride phosphate, glyceryl stearate SE, ceteth-20, triethyl citrate, PEG-20 methylglucose sesquistearate, ceteareth-12, glyceryl stearate citrate, cetyl phosphate, triceteareth-4 phosphate, trilaureth-4 phosphate, polyglyceryl methylglucose distearate, potassium cetyl phosphate, isosteareth-10, polyglyceryl-2 sesquiisostearate, ceteth-10, oleth-20, isoceteth-20, glyceryl stearate in a mixture with ceteareth-20, ceteareth-12, cetylstearyl alcohol and cetyl palmitate, cetylstearyl alcohol in a mixture with PEG-20 stearate, PEG-30 stearate, PEG-40 stearate, and PEG-100 stearate.

In yet additional embodiments in which the oil phase of the preparations consists at least partially of silicone oils, silicone emulsifiers find use. In some embodiments, the silicone emulsifiers are selected from the group of interface-active substances, alkylmethicone copolyols, and/or alkyl dimethicone copolyols, particularly from the group of compounds characterized by the following chemical structure:

in which X and Y, independently of one another, are chosen from the group H and the branched and unbranched alkyl groups, acyl groups and alkoxy groups having 1 to 24 carbon atoms, p is a number from 0 to 200, q is a number from 1 to 40, and r is a number from 1 to 100. Some examples of silicone emulsifiers which find use in the present invention include, but are not limited to dimethicone copolyols (e.g., ABIL® B 8842, ABIL® B 8843, ABIL® B 8847, ABIL® B 8851, ABIL® B 8852, ABIL® B 8863, ABIL® B 8873, and ABIL® B 88183, all of which are commercially available from Th. Goldschmidt AG). An additional example of an interface-active substances which finds use in the present invention includes cetyldimethicone copolyol (ABIL® EM 90), as well as cyclomethiconedimethicone copolyol (ABIL® EM 97), both of which are commercially available from Th. Goldschmidt AG. An additional emulsifier which has proven useful in various compositions that finds use in embodiments of the present invention is laurylmethicone copolyol (Dow Corning® 5200 Formulation Aid), which is commercially available from Dow Corning Ltd.

In preferred embodiments of the present invention, the total amount of emulsifiers used in the personal care compositions (e.g., cosmetic or skin care preparations) are present in the range from about 0.1 to about 10.0% by weight, preferably about 0.5 to about 5.0% by weight, based on the total weight of the preparations. However, it is not intended that the present invention be limited to any specific concentration of emulsifier and/or co-emulsifier, as various embodiments of the present invention have different preferred concentrations and/or concentration ranges.

In some embodiments, the present invention provides emulsions in various forms, including skin protection creams, skin lotions, cosmetic milks, sunscreen creams, and sun protection milks. In some preferred embodiments, these compositions comprise fats, oils, waxes, and/or other fatty substances, as well as water, and one or more emulsifiers as are customarily used for such a type of formulation.

In addition to the liquid and somewhat more solid emulsions of the cosmetic cleansing lotions and/or cleansing creams of the present invention, the present invention also provides sprayable cleansing preparations (“cleansing sprays”), which are used, for example, for removing make-up or as mild washing lotion. In addition, these cleansing sprays find use in applications for treatment of blemished skin. These cleansing preparations also find use as “rinse-off preparations” (i.e., products which are rinsed off the skin following application).

In addition to the above constituents, various embodiments of the present invention include additional components, such as auxiliaries and additives, including but not limited to bodying agents, fillers, perfume, dyes, emulsifiers, additional active ingredients (e.g., vitamins and proteins), light protection agents, stabilizers, insect repellents, alcohol, self-tanning substances, water, salts, antimicrobials, proteases, and/or keratinase, etc. Indeed, it is not intended that the present invention be limited to any particular components, as long as the active component comprising a scaffold and a peptide is included. It is further contemplated that the present invention will find use in numerous and various medicinal preparations.

In some embodiments, the compositions of the present invention contain an emulsifier and/or surfactant, generally to help disperse and suspend the disperse phase within the continuous aqueous phase. A surfactant may also be useful if the product is intended for skin or hair cleansing. For convenience hereinafter, “emulsifiers” are encompassed by the term “surfactants.” Thus, as used herein, the term “surfactant(s)” refers to surface active agents, whether used as emulsifiers or for other surfactant purposes such as skin cleansing. Known, including conventional surfactants find use in the present invention, provided that the selected agent is chemically and physically compatible with essential components of the composition and provides the desired characteristics (See e.g., WO 00/24372). Suitable surfactants include non-silicone derived materials, silicone-derived materials, and mixtures thereof.

In further embodiments, the compositions of the present invention comprise preferably from about 0.05% to about 30%, more preferably from about 0.5% to 15%, and most preferably from about 1% to 10% of a surfactant or mixture of surfactants. The exact surfactant or surfactant mixture chosen depends upon the pH of the composition, the other components present and the desired final product aesthetics.

Among the nonionic surfactants that are useful herein are those that can be broadly defined as condensation products of long chain alcohols (e.g., C₈₋₃₀ alcohols), with sugar or starch polymers (e.g., glycosides). Other useful nonionic surfactants include the condensation products of alkylene oxides with fatty acids (i.e., alkylene oxide esters of fatty acids). These materials have the general formula RCO(X)_(n)OH wherein R is a C₁₀₋₃₀ alkyl group, X is —OCH₂CH₂— (i.e., derived from ethylene glycol or oxide) or —OCH₂CHCH₃— (i.e., derived from propylene glycol or oxide) and n is an integer from about 6 to about 200. Other nonionic surfactants are the condensation products of alkylene oxides with 2 moles of fatty acids (i.e., alkylene oxide diesters of fatty acids). These materials have the general formula RCO(X)_(n)OOCR wherein R is a C₁₀₋₃₀ alkyl group, X is —OCH₂CH₂— (i.e., derived from ethylene glycol or oxide) or —OCH₂CHCH₃— (i.e., derived from propylene glycol or oxide) and n is an integer from about 6 to about 100. In some embodiments, an emulsifier for use herein is preferably a fatty acid ester blend based on a mixture of sorbitan fatty acid ester and sucrose fatty acid ester, especially a blend of sorbitan stearate and sucrose cocoate. Further suitable examples include a mixture of cetearyl alcohols and cetearyl glucosides. However, it is not intended that the present invention be limited to any particular emulsifier, as various suitable emulsifiers are known in the art.

In additional embodiments, the hydrophilic surfactants useful herein alternatively or additionally include any of a wide variety of cationic, anionic, zwitterionic, and amphoteric surfactants such as are known in the art (See, e.g., McCutcheon's, Emulsifiers and Detergents, North American and International Editions, MC Publishing Co. [2003]; U.S. Pat. No. 5,011,681 U.S. Pat. No. 4,421,769; and U.S. Pat. No. 3,755,560).

In some additional embodiments, interface- and/or surface-active agents are included in some personal care compositions of the present invention, including but not limited to cationic emulsifiers (e.g., quaternary surfactants).

Quaternary surfactants that contain at least one N atom which is covalently bonded to 4 alkyl or aryl groups. This leads, irrespective of the pH, to a positive charge. Alkylbetain, alkylamidopropylbetain and alkylamidopropylhydroxysultaine are examples of quaternary surfactants that find use in some embodiments of the present invention.

The cationic surfactants provided in some embodiments of the present invention also include, but are not limited to quaternary ammonium compounds, in particular benzyltrialkylammonium chlorides or bromides (e.g., benzyldimethylstearylammonium chloride), alkyltrialkylammonium salts (e.g., cetyltrimethylammonium chloride or bromide), alkyldimethylhydroxyethylammonium chlorides or bromides, dialkyldimethylammonium chlorides or bromides, alkylamidoethyltrimethylammonium ether sulfates, alkylpyridinium salts (e.g., lauryl- or cetylpyrimidinium chloride), imidazoline derivatives, and compounds with a cationic character, such as amine oxides (e.g., alkyldimethylamine oxides or alkylaminoethyldimethylamine oxides). In some preferred embodiments, cetyltrimethylammonium salts find use in some personal care compositions of the present invention.

In yet additional embodiments, cationic polymers (e.g., JAGUAR® C 162 [hydroxypropyl guar hydroxypropyltrimonium chloride]), modified magnesium aluminum silicates (e.g., quaternium-18-hectorite, which is commercially available (e.g., BENTONE® 38; Rheox), and/or stearalkonium hectorite, which is commercially available (e.g., SOFTISAN® gel; Hüls AG) find use in some personal care compositions of the present invention. However, it is not intended that the present invention be limited to any particular cationic polymer.

In some yet further embodiments, some compositions of the present invention comprise oil thickeners in order to improve the tactile properties of emulsions. Preferred oil thickeners include, but are not limited to other solids (e.g., hydrophobic silicon oxides of the AEROSIL® type, which are available from Degussa AG). Examples of advantageous AEROSIL® oxide grades include AEROSIL® OX50, AEROSIL® 130, AEROSIL® 150, AEROSIL® 200, AEROSIL® 300, AEROSIL® 380, AEROSIL® MOX 80, AEROSIL® MOX 170, AEROSIL® COK 84, AEROSIL® R 202, AEROSIL® R 805, AEROSIL® R 812, AEROSIL® R 972, AEROSIL® □R 974 and AEROSIL® R976.

In some additional embodiments, some personal care compositions of the present invention comprise at least one “metal soap” (i.e., a salt of a higher fatty acid, with the exception of alkali metal salt), which are function as oil thickeners. Examples of such metal soaps include, but are not limited to aluminum stearate, zinc stearate and/or magnesium stearate.

A variety of anionic surfactants are also useful herein (See e.g., U.S. Pat. No. 3,929,678). Exemplary anionic surfactants include, but are not limited to alkoyl isethionates (e.g., C₁₂-C₃₀), alkyl and alkyl ether sulfates and salts thereof, alkyl and alkyl ether phosphates and salts thereof, alkyl methyl taurates (e.g., C₁₂-C₃₀), and soaps (e.g., substituted alkylamine and alkali metal salts, e.g., sodium or potassium salts) of fatty acids.

Amphoteric and zwitterionic surfactants are also useful herein. Examples of preferred amphoteric and zwitterionic surfactants which find use in the compositions of the present invention are those which are broadly described as derivatives of aliphatic secondary and tertiary amines in which the aliphatic radical can be straight or branched chain and wherein one of the aliphatic substituents contains from about 8 to about 22 carbon atoms (preferably C₈-C₁₈) and one contains an anionic water solubilizing group (e.g., carboxy, sulfonate, sulfate, phosphate, or phosphonate). Examples, include but are not limited to alkyl imino acetates and iminodialkanoates and aminoalkanoates, imidazolinium and ammonium derivatives. Other suitable amphoteric and zwitterionic surfactants are those selected from the group consisting of betaines, sultaines, hydroxysultaines, and branched and unbranched alkanoyl sarcosinates, and mixtures thereof.

In some further embodiments, some personal care compositions comprise at least one amphoteric and/or zwitterionic surfactant (e.g., cocamidopropylbetain) and/or moisturizer (e.g. betain). Examples of amphoteric surfactants that find use in such embodiments of the present invention include but are not limited to acyl/dialkylethylenediamine (e.g., sodium acylamphoacetate), disodium acylamphodipropionate, disodium alkylamphodiacetate, sodium acylamphohydroxypropylsulfonate, disodium acylamphodiacetate, sodium acylamphopropionate, N-alkylamino acids, for example aminopropylalkylglutamide, alkylaminopropionic acid, sodium alkylimidodipropionate, and lauroamphocarboxyglycinate.

In some embodiments, the amount of surface- or interface-active substances (one or more compounds) in the preparations is preferably between about 0.001 and about 30% by weight, and more preferably between about 0.05 and about 20% by weight, in most preferably between about 1 and about 10% by weight, based on the total weight of the preparation.

In some yet additional embodiments, the active ingredients (one or more compounds) comprise at least one lipophilic active ingredient. In some embodiments, these lipophilic active ingredients are selected from the group consisting of acetylsalicylic acid, atropine, azulene, hydrocortisone and derivatives thereof (e.g., hydrocortisone-17-valerate), B vitamins, D vitamins, vitamin B₁, vitamin B₁₂, vitamin D₁, retinoid, bisabolol, unsaturated fatty acids (e.g., the essential fatty acids often also referred to as “vitamin F”), γ-linolenic acid, oleic acid, eicosapentenoic acid, docosahexenoic acid and derivatives thereof, chloramphenicol, caffeine, prostaglandins, thymol, camphor, extracts or other products of a vegetable and animal origin (e.g. evening primrose oil, borrage oil or currant seed oil, fish oils, cod-liver oil), and ceramides and ceramide-like compounds, etc. In some embodiments, the active ingredient(s) are refatting substances (e.g., purcellin oil, EUCERIT® and/or NEROCERIT®).

In further embodiments, some emulsions of the present invention include a silicone containing emulsifier or surfactant. A wide variety of silicone emulsifiers find use herein. These silicone emulsifiers are typically organically modified organopolysiloxanes, also known to those skilled in the art as silicone surfactants. Useful silicone emulsifiers include, but are not limited to dimethicone copolyols. These materials are polydimethyl siloxanes which have been modified to include polyether side chains such as polyethylene oxide chains, polypropylene oxide chains, mixtures of these chains and polyether chains containing moieties derived from both ethylene oxide and propylene oxide. Other examples include alkyl-modified dimethicone copolyols (i.e., compounds which contain C₂-C₃₀ pendant side chains). Still other useful dimethicone copolyols include materials having various cationic, anionic, amphoteric, and zwitterionic pendant moieties.

In some embodiments, the compositions of the present invention comprise at least one polymeric thickening agent. The polymeric thickening agents useful herein preferably have a number average molecular weight of greater than about 20,000, more preferably greater than about 50,000, and most preferably greater than about 100,000. In some embodiments, the compositions of the present invention comprise from about 0.01% to about 10%, preferably from about 0.1% to about 8% and more preferably from about 0.2% to about 5% by weight of the composition of the polymeric thickening agent or mixtures thereof.

Preferred polymer thickening agents for use herein include, but are not limited to non-ionic thickening agents and anionic thickening agents or mixtures thereof. Suitable non-ionic thickening agents include, but are not limited to polyacrylamide polymers, crosslinked poly(N-vinylpyrrolidones), polysaccharides, natural or synthetic gums, polyvinylpyrrolidone and polyvinylalcohol. Suitable anionic thickening agents include, but are not limited to acrylic acid/ethyl acrylate copolymers, carboxyvinyl polymers and crosslinked copolymers of alkyl vinyl ethers and maleic anhydride. Commercially available thickeners (e.g., Carbopol; Noveon) find use in some embodiments of the present invention. Suitable Carbopol resins may be hydrophobically modified, and other suitable resins are described in WO98/22085, or mixtures thereof.

In some embodiments of the present invention, the water phase has a gel character which, in addition to an effective content of compounds and solvents (as appropriate) preferably comprises water, further organic and/or inorganic thickeners, and/or hydrocolloids.

In some embodiments, inorganic thickeners are selected from the group consisting of modified, unmodified, naturally occurring, and synthetic phyllosilicates. Although it is generally preferable to use pure components, in some embodiments, mixtures of different modified and/or unmodified phyllosilicates find use in various compositions of the present invention. As generally known in the art, phyllosilicates are silicates and alumosilicates in which the silicate or aluminate units are linked together via three Si—O— or Al—O— bonds and form a wavy sheet or layer structure. The fourth Si—O— or Al—O— valence is saturated by cations. Relatively weak electrostatic interactions (e.g. hydrogen bridge bonds), exist between the individual layers. The layer structure is largely defined by strong, covalent bonds. The stochiometry of the sheet silicates is (Si₂O₅ ²⁻) for pure silicate structures and (Al_(m)Si²⁻ _(m)O₅ ^((2+m)−)) for alumosilicates, wherein “m” is a number greater than zero and less than 2. In some embodiments in which alumosilicates are present in the absence of pure silicates, each Si⁴⁺ group replaced by Al³⁺ requires another singly charged cation to neutralize the charge. The charge balance is preferably evened out by H⁺, alkali metal ions or alkaline earth metal ions. In alternative embodiments, aluminum is used as a counterion. In contrast to the alumosilicates, these compounds are referred to as “aluminum silicates.” “Aluminum alumosilicates,” in which aluminum is present both in the silicate network, and also as counterion, also find use in some embodiments of the present invention.

Phyllosilicates are well known in the art (See e.g., Hollemann et al., Lehrbuch der Anorganischen Chemie [Textbook of Inorganic Chemistry], 91 st-100th Ed., Walter de Gruyter—Verlag [1985]; Remy, Lehrbuch der Anorganischen Chemie, 12^(th) Ed., Akademische Verlagsgesellschaft, Leipzig [1965]). The layer structure of montmorillonite is also known (See, Römpps Chemie-Lexikon, Franckh'sche Verlagshandlung, W. Keller & Co., Stuttgart, 8^(th) Ed., [1985], p. 2668 f). Examples of phyllosilicates include the following (montmorillonite is the main mineral comprising the naturally-occurring bentonites);

Montmorillonite Na_(0.33)((Al_(1.67)Mg_(0.33))(OH)₂(S_(i4)O₁₀)) often simplified: Al₂O₃*4SiO₂*H₂O*nH₂O or Al₂[(OH)₂/Si₄O₁₀]•nH₂O Kaolinite Al₂(OH)₄(Si₂O₅) Illite (K,H₃O)_(y)(Mg₃(OH)₂(Si_(4-y)Al_(y)O₁₀)) or (K,H₃O)_(y)(Al₂(OH)₂(Si_(4-y)Al_(y)O₁₀)) where y = 0.7-0.9 Beidellite (Ca,Na)_(0.3)(Al₂(OH)₂(Al_(0.5)Si_(3.5)O₁₀)) Nontronite Na_(0.33)(Fe₂(OH)₂(Al_(0.33)S_(i3.67)O₁₀)) Saponite (Ca,Na)_(0.33)((Mg,Fe)₃(OH)₂(Al_(0.33)Si_(3.67)O₁₀)) Hectorite Na_(0.33)((Mg,Li)₃(OH,F)₂(Si₄O₁₀))

In some preferred embodiments, inorganic gel formers including but not limited to aluminum silicates, such as the montmorillonites (bentonites, hectorites and derivatives thereof, such as quaternium-18 bentonite, quaternium-18 hectorites, stearalkonium bentonites and stearalkonium hectorites), and also magnesium-aluminum silicates (VEEGUM® grades), and sodium-magnesium silicates (LAPONITE® grades) find use in the present invention.

Montmorillonites represent clay minerals which are a type of dioctahedral smectites, and are masses which swell in water, but do not become plastic. The layer packets in the three-layer structure of the montmorillonites can swell as a result of reversible incorporation of water (in a 2- to 7-fold amount) and other substances, such as, for example, alcohols, glycols, pyridine, picoline, ammonium compounds, hydroxy-aluminosilicate ions etc. The chemical formula given above provides just an approximation of the formula, as montmorillonites have a large capacity for ion exchange. For example, Al can be replaced by Mg, Fe²⁺, Fe³⁺, Zn, Pb (e.g., from harmful substances in waste waters), Cr, Cu and other elements. The resulting negative charge of the octahedral layers is compensated for by the presence of cations, in particular Na⁺ (i.e., sodium montmorillonite) and Ca²⁺ (i.e., calcium montmorillonite, a compound that is only swellable to a very small extent) in interlayer positions.

In alternative embodiments, synthetic magnesium silicates and/or bentonites find use in the present invention, including but not limited to such commercially available compounds as OPTIGEL® (Süd-Chemie). As indicated above, in some embodiments, aluminum silicates such as the commercially available VEEGUM® (R.T. Vanderbilt Comp., Inc), find use in the present invention. Various VEEGUM® grades which find use in various embodiments of the present invention are provided below.

VEEGUM ® Grades Regular Grade HV K HS S-728 SiO₂ 55.5 56.9 64.7 69.0 65.3 MgO 13.0 13.0 5.4 2.9 3.3 Al₂O₃ 8.9 10.3 14.8 14.7 17.0 Fe₂O₃ 1.0 0.8 1.5 1.8 0.7 CaO 2.0 2.0 1.1 1.3 1.3 Na₂O 2.1 2.8 2.2 2.2 3.8 K₂O 1.3 1.3 1.9 0.4 0.2 Ashing loss 11.1 12.6 7.6 5.5 7.5

The above products swell in water to form viscous gels, which have an alkaline reaction. The organophilization of montmorillonite or bentonites (exchange of the interlayer cations for quaternary alkylammonium ions) produces products (bentones) which are preferably used for dispersion in organic solvents and oils, fats, ointments, inks, surface coatings and in detergents.

BENTONE® is a trade name for various neutral and chemically inert gelling agents which are constructed from long-chain, organic ammonium salts and specific types of montmorillonite. BENTONE® gelling agents swell in organic media, which cause the media to also swell. The gels are resistant to diluted acids and alkalis, although they partially lose their gelling properties upon prolonged contact with strong acids and alkalis. Because of their organophilic character, BENTONE® gelling agents are only wettable by water with difficulty. There are various BENTONE® gelling agent grades commercially available, including those sold by Kronos Titan: BENTONE® 27, an organically modified montmorillonite; BENTONE® 34 (dimethyldioctylammonium bentonite; prepared in accordance with U.S. Pat. No. 2,531,427, incorporated herein by reference, which because of its lipophilic groups, swells more readily in lipophilic medium than in water); BENTONE® 38, an organically modified montmorillonite, available as a cream-colored to white powder; BENTONE® LT, a purified clay mineral; BENTONE® Gel MIO, an organically modified montmorillonite which is supplied as a very fine suspension in mineral oil (SUS-71) (10% bentonite, 86.7% mineral oil and 3.3% wetting agent); BENTONE® Gel IPM, an organically modified bentonite which is suspended in isopropyl myristate (10% bentonite, 86.7% isopropylmyristate, 3.3% wetting agent); BENTONE® Gel CAO, an organically modified montmorillonite which is taken up in castor oil (10% bentonite, 86.7% castor oil and 3.3% wetting agent); BENTONE® Gel Lantrol, an organically modified montmorillonite which, in paste form, is intended for the further processing, in particular for the preparation, of cosmetic compositions; 10% bentonite, 64.9 LANTROL® (wool wax oil), 22.0 isopropyl myristate, 3.0 wetting agent and 0.1 propyl p-hydroxybenzoate; BENTONE® Gel Lan I, a 10% strength BENTONE® 27 paste in a mixture of wool wax USP and isopropyl palmitate; BENTONE® Gel Lan II, a bentonite paste in pure liquid wool wax; BENTONE® Gel NV, a 15% strength BENTONE® 27 paste in dibutyl phthalate; BENTONE® Gel OMS, a bentonite paste in Shellsol T.; BENTONE® Gel OMS 25, a bentonite paste in isoparaffinic hydrocarbons (IDOPAR® H); and BENTONE® Gel IPP, a bentonite paste in isopropyl palmitate.

“Hydrocolloid” is the technological abbreviation for the more correct name “hydrophilic colloid.” Hydrocolloids are macromolecules which have a largely linear structure and intermolecular forces of interaction which permit secondary and primary valence bonds between the individual molecules to form a recticular structure. Some hydrocolloids are water-soluble natural or synthetic polymers which, in aqueous systems, form gels or viscous solutions. These compounds increase the viscosity of water by either binding water molecules (hydration), or by absorbing and encapsulating the water into their interwoven macromolecules, while restricting the mobility of water. These water-soluble polymers represent a large group of natural and synthetic polymers that are chemically very different, but which share a common feature in their solubility in water or aqueous media. A prerequisite for this is that these polymers have a number of hydrophilic groups sufficient for solubility in water and are not too greatly crosslinked. These hydrophilic groups can be nonionic, anionic or cationic in nature, for example as follows:

In some preferred embodiments, the group of the cosmetically and dermatologically relevant hydrocolloids are divided into the following groups: organic, natural compounds (e.g., agar agar, carrageen, tragacanth, gum arabic, alginates, pectins, polyoses, guar flour, carob bean flour, starch, dextrins, gelatins, and casein); organic, modified natural substances (e.g., carboxymethylcellulose and other cellulose ethers, hydroxyethylcellulose and hydroxypropylcellulose and microcristalline cellulose); organic, completely synthetic compounds (e.g., polyacrylic and polymethacrylic compounds, vinyl polymers, polycarboxylic acids, polyethers, polyimines, polyamides, and polyurethanes); and inorganic compounds (e.g., polysilicic acids, clay minerals, such as montmorillonites, zeolites, and silicas).

In alternative embodiments, ethylcelluloses find use in compositions of the present invention as stabilizers. Ethylcelluloses are characterized by the following structure. In this structure, the Rs are either ethyl groups or hydrogen atoms.

In some preferred embodiments, the degree of ethylation in the ethylcellulose is from about 2.0 to about 3.0, corresponding to about 40 to about 55%, and more preferably about 48.0 to about 49.5% ethylation. The average molecular mass is preferably chosen such that the viscosity of an approximately 5% strength solution in a mixture of 80 parts of toluene and 20 parts of ethanol at 25° C. is 3 to 110 mPas, and more preferably 9 to 11 mPas. In some particularly preferred embodiments, the average molar mass is from about 100,000 to about 400,000 g/mol. In some preferred embodiments, the ethylcellulose concentration in compositions of the present invention ranges from about 0.1 to about 10% by weight, based on the total weight of the preparations. Various ethylcelluloses find use in the present invention, including but not limited to those that are commercially available (e.g., ETHOCEL® Standard 10 Premium; Dow Chemicals).

In yet additional embodiments, microcristalline cellulose finds use as hydrocolloid in compositions of the present invention. Various microcrystalline cellulose preparations find use in the present invention, including but not limited to those that are commercially available (e.g., AVICEL®, such as AVICEL® RC-591, as well as AVICEL® RC/CL; AVICEL® CE-15; and AVICEL® 500; FMC Corporation Food and Pharmaceutical Products). In some particularly preferred embodiments, AVICEL® RC-591 (a modified microcristalline cellulose which is made up of 89% microcrystalline cellulose and 11% sodium carboxymethylcellulose) finds use in the present invention.

Additional hydrocolloids that find use in the present invention include methylcelluloses (i.e., methylesters of cellulose). These compounds are characterized by the following structural formula

in which R can be a hydrogen or a methyl group.

Cellulose mixed ethers (generally referred to as methylcelluloses, which contain, in addition to a predominating content of methyl groups, also 2-hydroxyethyl, 2-hydroxypropyl or 2-hydroxybutyl groups) also find use in some embodiments of the present invention. In some preferred embodiments, hydroxypropyl)methyl-celluloses (e.g., METHOCEL® E4M; Dow Chemical Co.) find use in the present invention.

In yet further embodiments sodium carboxymethylcellulose (i.e., the sodium salt of the glycolic ether of cellulose, for which R in the above structural formula may be hydrogen and/or CH₂—COONa) finds use in the present invention. In some preferred embodiments, sodium carboxymethylcellulose, also sometimes referred to as “cellulose gum” (e.g., NATROSOL® Plus 330 CS; Aqualon) finds use in the present invention.

In additional embodiments, xanthan (CAS No. 11138-66-2), (i.e., xanthan gum), an anionic heteropolysaccharide generally formed by fermentation from maize sugar and isolated as potassium salt finds use in the present invention. It is produced by Xanthomonas campestris and some other species under aerobic conditions and has a molecular weight of from 2×10⁶ to 24×10⁶. Xanthan is formed from a chain having cellulose with side chains. The structure of the subgroups consists of glucose, mannose, glucuronic acid, acetate and pyruvate. The number of pyruvate units determines the viscosity of the xanthan.

In still further embodiments, carrageen is used as a gel former in compositions of the present invention. This compound is an extract from North Atlantic red algae (Florideae; Chondrus crispus and Gigartina stellata) that has a structure similar to that of agar. The term “carrageen” is frequently used in reference to a dried algae product and “carrageenan” is used in reference to the extract thereof. The carrageen precipitated from the hot-water extract of the algae is a colorless to sand-colored powder with a molecular weight range from about 100,000 to about 800,000 and a sulfate content of about 25%. Carrageen, which is very readily soluble in warm water, forms a thixotropic gel upon cooling, even if the water content is 95-98%. The rigidity of the gel is effected by the double helix structure of the carrageen.

In the case of carrageenan, three principle constituents are differentiated. The gel-forming “κ fraction” consists of D-galactose 4-sulfate and 3,6-anhydro-α-D-galactose, which has alternate glycoside bonds in the 1,3- and 1,4 position (in contrast, agar contains 3,6-anhydro-α-L-galactose). The nongelling “λ fraction” is composed of 1,3-glycosidically linked D-galactose 2-sulfate and 1,4-bonded D-galactose-2,6-disulfate radicals, and is readily soluble in cold water. Finally, “ι-carrageenan,” composed of D-galactose 4-sulfate in 1,3 bond and 3,6-anhydro-α-D-galactose 2-sulfate in 1,4 bond, is both water-soluble and also gel-forming. The nature of any cations which are present (K⁺, NH₄ ⁺, Na⁺, Mg²⁺, Ca²⁺) also influences the solubility of the carrageens.

In yet additional embodiments, chitosan (i.e., partially deacylated chitin) finds use in various compositions of the present invention. Chitosan has film-forming properties and is characterized as having a silky feel on the skin. One disadvantage for some uses, is its severe stickiness on the skin which occurs in temporarily (usually) during application. Due to this stickiness, some preparations are not acceptable to consumers. However, chitosan finds use in some preparations, including hair care compositions, as it is better than chitin in thickening and/or stabilizing, as well as improving the adhesion and water resistance of polymeric films. The use of chitosan is well-known to those of skill in the personal care art (See e.g., Fiedler, Lexikon der Hilfsstoffe für Pharmazie, Kosmetik und angrenzende Gebiete, [Lexikon of auxiliaries for pharmacy, cosmetics and related fields], 3^(rd) edition, Editio Cantor, Aulendorf, [1989], p. 293). Chitosan is characterized by the following structural formula:

where n assumes values up to about 10 000, and X is either the acetyl radical or hydrogen. Chitosan forms by deacetylation and partial depolymerization (hydrolysis) of chitin, which is characterized by the structural formula

Chitin is an essential constituent of the arthropod (e.g. insects, crabs, and spiders) ectoskeleton, and is also found in the connective and/or supporting tissues of other organisms (e.g. mollusks, algae, and fungi). In the region of about pH <6, chitosan is positively charged and in that range is also soluble in aqueous systems. It is incompatible with anionic raw materials. For this reason, in order to prepare chitosan-containing oil-in-water emulsions, the use of nonionic emulsifiers is appropriate (See e.g., EP 776 657). In some preferred embodiments, the compositions of the present invention contain at least one chitosans with a degree of deacetylation of at least about >25%, and more preferably, a range of more than about 55 to about 99% (as determined by means of ¹H-NMR). In some embodiments, chitosans of molecular weights between about 10,000 and about 1,000,000, in particular those with molecular weights between 100,000 and 1,000,000 (determined by means of gel permeation chromatography) find use in the present invention.

In yet further embodiments, polyacrylates find use as gelling agents in some compositions of the present invention. Suitable polyacrylates include but are not limited to acrylate-alkyl acrylate copolymers, in particular those chosen from the group of carbomers or CARBOPOL® copolymers (B. F. Goodrich Co.). In particular, the acrylate-alkyl acrylate copolymers that find use in some embodiments of the present invention have the following structure:

where R′ is a long-chain alkyl radical, and x and y represent numbers which symbolize the respective stoichiometric proportion of each of the comonomers.

In some embodiments, acrylate copolymers and/or acrylate-alkyl acrylate copolymers, include but are not limited to those that are commercially available (e.g., CARBOPOL® 1382, CARBOPOL® 981, and CARBOPOL® 5984; B. F. Goodrich Co., and in particular, polyacrylates from the group of CARBOPOL grades 980, 981, 1382, 2984, 5984 and Carbomer 2001). In additional embodiments, copolymers of C₁₀₋₃₀-alkyl acrylates and one or more monomers of acrylic acid, of methacrylic acid or esters thereof which are crosslinked with an allyl ether of sucrose or an allyl ether of pentaerythrito find use in some embodiments of the present invention.

Compounds which carry the INCI name “Acrylates/C₁₀₋₃₀ Alkyl Acrylate Crosspolymer” also find use in some embodiments of the present invention. In some embodiments, commercially available polymers (e.g., PEMULEN® TR1 and PEMULEN® TR2; B. F. Goodrich Co.) find use in some embodiments of the present invention, although it is not intended that the present invention be limited to any specific acrylate-containing composition.

In yet additional embodiments, compounds which carry the INCI name “ammonium acryloyldimethyltaurates/vinylpyrrolidone copolymers” find use in the present invention. These ammonium acryloyldimethyl taurate/vinylpyrrolidone copolymers have the empirical formula [C₇H₁₆N₂SO₄]_(n)[C₆H₉NO]_(m), which corresponds to the following structure:

Preferred species of this compound are listed in Chemical Abstracts under the Registry numbers 58374-69-9, 13162-05-5 and 88-12-0, and are commercially available (e.g., ARISTOFLEX®; Clariant GmbH). However, it is not intended that the present invention be limited to any particular species. In yet additional embodiments of the present invention, copolymers/crosspolymers comprising acryloyldimethyl taurate (e.g., SIMUGEL® EG and SIMUGEL® EG; Seppic S.A.) find use in some compositions of the present invention.

Additional completely synthetic hydrocolloids that find use in the present invention include, but are not limited to anionic polyurethanes which are soluble or dispersible in water and which are advantageously obtainable from:

-   -   Aa) at least one compound which contains two or more active         hydrogen atoms per molecule,     -   Ab) at least one diol containing acid or salt groups, and     -   Ac) at least one diisocyanate.

In some preferred embodiments, the component Aa) is, in particular, a diol, aminoalcohol, diamine, polyesterol, polyetherol with a number-average molecular weight of in each case up to 3000, or mixtures thereof, where up to 3 mol % of said compounds may be replaced by triols or triamines. Preference is given to diols and polyesterdiols. In particular, the component Aa) comprises at least 50% by weight, based on the total weight of the component Aa), of a polyesterdiol. Suitable polyesterdiols are all those which are customarily used for the preparation of polyurethanes, in particular the reaction products of phthalic acid and diethylene glycol, isophthalic acid and 1,4-butanediol, isophthalic acid/adipic acid and 1,6-hexanediol, and adipic acid and ethylene glycol or 5-NaSO₃-isophthalic acid, phthalic acid, adipic acid and 1,6-hexanediol.

Examples of diols which find use in some embodiments of the present invention include, but are not limited to ethylene glycol, propylene glycol, butylene glycol, neopentyl glycol, polyetherols (e.g., polyethylene glycols having molecular weights up to 3000), block copolymers of ethylene oxide and propylene oxide with number-average molecular weights of up to 3000, and block copolymers of ethylene oxide, propylene oxide and butylene oxide which contain the copolymerized alkylene oxide units in randomly distributed manner or in the form of blocks. Preference is given to ethylene glycol, neopentyl glycol, di-, tri-, tetra-, penta- or hexaethylene glycol. Other diols which find use include poly(α-hydroxycarboxylic acid)diols.

Suitable aminoalcohols that find use in some embodiments of the present invention include but are not limited to 2-aminoethanol, 2-(N-methylamino)ethanol, 3-aminopropanol, and 4-aminobutanol.

In some embodiments, diamines such as ethylenediamine, propylenediamine, 1,4-diaminobutan, 1,6-diaminohexane, and α,ω-diamines which can be prepared by amination of polyalkylene oxides with ammonia find use in some compositions of the present invention.

Component Ab) is, in particular, dimethylolpropanoic acid or a compound with the formula:

where RR is in each case a C₂-C₁₈-alkylene group and Me is Na or K.

Component Ac) is, in particular, hexamethylene diisocyanate, isophorone diisocyanate, methyldiphenyl isocyanate (MDI), and/or tolylene diisocyanate.

In some embodiments, the polyurethanes are obtained by reacting the compounds of groups Aa) and Ab) under an inert-gas atmosphere in an inert solvent at temperatures of from 70 to 130° C. with the compounds of group Ac). This reaction can be carried out, where appropriate, in the presence of chain extenders in order to prepare polyurethanes with relatively high molecular weights. As is customary in the preparation of polyurethanes, the components [(Aa)+(Ab)]:Ac are advantageously used in the molar ratio of from 0.8 to 1.1:1. The acid number of the polyurethanes is determined by the composition and the concentration of the compounds of component (Ab) in the mixture of components (Aa) and (Ab).

In some embodiments, the polyurethanes have K values according to H. Fikentscher (determined in 0.1% strength by weight solutions in N-methylpyrrolidone at 25° C. and pH 7) of from about 15 to about 100, and preferably about 25 to about 50. The K value (i.e., “intrinsic viscosity”), is a parameter which is easy to determine by means of viscosity measurements of polymer solutions and is therefore frequently used in the industrial sector for characterizing polymers. Polyurethanes containing acid groups that find use in some embodiments of the present invention include, but are not limited to polyurethanes that are water-soluble or dispersible without the aid of emulsifiers after partial or complete neutralization. The salts of the polyurethanes generally have better solubility or dispersibility in water than the unneutralized polyurethanes. Bases which find use for the neutralization of the polyurethanes include alkali metal bases (e.g., sodium hydroxide solution, potassium hydroxide solution, soda, sodium hydrogencarbonate, potassium carbonate or potassium hydrogen carbonate) and alkaline earth metal bases (e.g., calcium hydroxide, calcium oxide, magnesium hydroxide or magnesium carbonate, and ammonia and amines). In some embodiments, 2-amino-2-methylpropanol, diethylaminopropylamine and triisoproanolamine find particular use in the neutralization of the polyurethanes containing acid groups. In yet additional embodiments, the neutralization of the polyurethanes containing acid groups is carried out using mixtures of two or more bases (e.g. mixtures of sodium hydroxide solution and triisopropanolamine). Depending on the intended use, neutralization is partial (e.g. about 20 to about 40%) or complete (i.e., 100%). These polymers and their preparation are described in more detail in DE-A-42 25 045, incorporated herein by reference.

B. Water-soluble or -dispersible cationic polyurethanes and polyureas of:

-   -   Ba) at least one diisocyanate, which may have already been         reacted beforehand with one or more compounds which contain two         or more active hydrogen atoms per molecule, and     -   Bb) at least one diol, primary or secondary amino alcohol,         primary or secondary diamine or primary or secondary triamine         with one or more tertiary, quaternary or protonated tertiary         amino nitrogen atoms.

Preferred diisocyanates are as given above under A). Compounds with two or more active hydrogen atoms are diols, aminoalcohols, diamines, polyesterols, polyamidediamines and polyetherols. Suitable compounds of this type are as given above under A).

The polyurethanes are prepared as described above under A). Charged cationic groups can be produced in the polyureas from the tertiary amino nitrogen atoms present either by protonation, (e.g., with carboxylic acids, such as lactic acid), or by quaternization (e.g. with alkylating agents, such as C₁ to C₄-alkyl halides) or sulfates. Examples of such alkylating agents include, but are not limited to ethyl chloride, ethyl bromide, methyl chloride, methyl bromide, dimethyl sulfate and diethyl sulfate. These polymers and their preparation are described in more detail in DE-A-42 41 118, which is incorporated herein by reference.

C. Linear polyurethanes with carboxylate groups of:

-   -   Ca) a 2,2-hydroxymethyl-substituted carboxylic acid of the         formula

-   -    in which RR′ is a hydrogen atom or a C₁-C₂₀-alkyl group, which         is used in an amount which suffices for about 0.35 to about 2.25         milliequivalents of carboxyl groups to be present in the         polyurethane per g of polyurethane,     -   Cb) about 10 to about 90% by weight, based on the weight of the         polyurethane, of one or more organic compounds with not more         than two active hydrogen atoms and     -   Cc) one or more organic diisocyanates.

In some preferred embodiments, the carboxyl groups present in the polyurethane are, finally, at least partially neutralized with a suitable base. These polymers and their preparation are described in EP-A-619 111, incorporated herein by reference.

D. Carboxyl-containing polycondensation products of anhydrides of tri- or tetracarboxylic acids and diols, diamines or aminoalcohols (polyesters, polyamides or polyester amides). These polymers and their preparation are described in more detail in DE-A-42 24 761, incorporated herein by reference.

E. Polyacrylates and polymethacrylates, as are described in more detail in DE-A-43 14 305, 36 27 970 and 29 17 504, all of which are incorporated herein by reference.

The polymers used in some embodiments of the present invention have a K value of from about 15 to about 100, and more preferably from about 25 to about 50. The polymers are generally present in the composition in an amount in the range from about 0.2 to about 20% by weight, based on the total weight of the compositions. The salt is used in an amount effective for improving the exchangeability of the polymers. The salt is generally used in an amount of from about 0.02 to about 10% by weight, and more preferably from about 0.05 to about 5% by weight, and in particular, from about 0.1 to about 3% by weight, based on the total weight of the composition.

The total amount of one or more hydrocolloids in some embodiments of the personal care compositions of the present invention is less than about 5% by weight, preferably between about 0.05 and about 3.0% by weight, and more preferably between about 0.1 and about 1.0% by weight, based on the total weight of the preparations.

In some embodiments, the present compositions comprise at least one silicone oil phase. Silicone oil phase(s) generally comprises from about 0.1% to about 20%, preferably from about 0.5% to about 10%, and more preferably from about 0.5% to about 5%, of the composition. The silicone oil phase preferably comprises one or more silicone components.

In some embodiments, silicone components are fluids, including straight chain, branched and cyclic silicones. Suitable silicone fluids useful herein include silicones inclusive of polyalkyl siloxane fluids, polyaryl siloxane fluids, cyclic and linear polyalkylsiloxanes, polyalkoxylated silicones, amino and quaternary ammonium modified silicones, polyalkylaryl siloxanes or a polyether siloxane copolymer and mixtures thereof. Volatile, as well as non-volatile silicone fluids find use herein. Silicone fluids generally have an average molecular weight of less than about 200,000. In preferred embodiments, suitable silicone fluids have a molecular weight of about 100,000 or less, preferably about 50,000 or less, and more preferably about 10,000 or less. Preferably the silicone fluid is selected from silicone fluids having a weight average molecular weight in the range from about 100 to about 50,000 and preferably from about 200 to about 40,000. Typically, silicone fluids have a viscosity ranging from about 0.65 to about 600,000 mm²s⁻¹, preferably from about 0.65 to about 10,000 mm²·s⁻¹ at 25° C. The viscosity can be measured by means of a glass capillary viscometer as set forth in Dow Corning Corporate Test Method CTM0004, Jul. 29, 1970. Suitable polydimethyl siloxanes that can be used herein include commercially available compounds (e.g., from the General Electric Company and Dow Corning). Also useful are essentially non-volatile polyalkylarylsiloxanes, for example, polymethylphenylsiloxanes, having viscosities of about 0.65 to 30,000 mm²s⁻¹ at 25° C. (General Electric Company or from Dow Corning). Cyclic polydimethylsiloxanes suitable for use herein are those having a ring structure incorporating from about 3 to about 7 (CH₃)₂SiO moieties, preferably about 5 or more.

In additional embodiments, silicone gums find use herein. In some preferred embodiments, a silicone oil phase comprises a silicone gum or a mixture of silicones including the silicone gum. Typically, silicone gums have a viscosity at 25° C. in excess of about 1,000,000 mm²s⁻¹. The silicone gums include dimethicones as known in the art (See e.g., U.S. Pat. No. 4,152,416; and Noll, Chemistry and Technology of Silicones, Academic Press, New York [1968]). Silicone gums such as those described in General Electric Silicone Rubber Product Data Sheets SE 30, SE 33, SE 54 and SE 76, also find use in the present invention. Specific examples of silicone gums include polydimethylsiloxane, (polydimethylsiloxane) (methylvinylsiloxane) copolymer, poly(dimethylsiloxane)(diphenyl)(methylvinylsiloxane) copolymer and mixtures thereof. Preferred silicone gums for use herein are silicone gums having a molecular weight of from about 200,000 to about 4,000,000 selected from dimethiconol, dimethicone copolyol, dimethicone and mixtures thereof.

In some embodiments, a silicone phase herein preferably comprises a silicone gum incorporated into the composition as part of a silicone gum-fluid blend. When the silicone gum is incorporated as part of a silicone gum-fluid blend, the silicone gum preferably constitutes from about 5% to about 40%, especially from about 10% to 20% by weight of the silicone gum-fluid blend. Suitable silicone gum-fluid blends herein are mixtures consisting essentially of:

(i) a silicone having a molecular weight of from about 200,000 to about 4,000,000 selected from dimethiconol, fluorosilicone and dimethicone and mixtures thereof; and

(ii) a carrier which is a silicone fluid, the carrier having a viscosity from about 0.65 mm²s⁻¹ to about 100 mm²s⁻¹,

wherein the ratio of i) to ii) is from about 10:90 to about 20:80 and wherein said silicone gum-based component has a final viscosity of from about 100 mm²s⁻¹ to about 100,000 mm²s⁻¹, preferably from 500 mm²s⁻¹ to about 10,000 mm²s⁻¹.

Further silicone components suitable for use in a silicone oil phase herein include crosslinked polyorganosiloxane polymers, optionally dispersed in a fluid carrier. In general, when present the crosslinked polyorganosiloxane polymers, together with its carrier (if present) comprises from about 0.1% to about 20%, preferably from about 0.5% to about 10%, and more preferably from about 0.5% to about 5% of the composition. Such polymers comprise polyorganosiloxane polymers crosslinked by a crosslinking agent (See e.g., WO98/22085). Examples of suitable polyorganosiloxane polymers for use herein include, but are not limited to methyl vinyl dimethicone, methyl vinyl diphenyl dimethicone and methyl vinyl phenyl methyl diphenyl dimethicone.

Another class of silicone components suitable for use in a silicone oil phase herein includes polydiorganosiloxane-polyoxyalkylene copolymers containing at least one polydiorganosiloxane segment and at least one polyoxyalkylene segment (See e.g., WO98/22085). Suitable polydiorganosiloxane-polyalkylene copolymers are available commercially under the tradenames BELSIL® from Wacker-Chemie GmbH. A particularly preferred copolymer fluid blend for use herein includes Dow Corning DC3225C which has the CTFA designation Dimethicone/Dimethicone copolyol.

In further embodiments, compositions of the present invention comprise an organic sunscreen. In some embodiments, suitable sunscreens have UVA absorbing properties, while others have UVB absorbing properties, and still others comprise a mixture thereof. The exact amount of the sunscreen active varies, depending upon the desired Sun Protection Factor (i.e., the “SPF”) of the composition, as well as the desired level of UV protection. SPF is a commonly used measure of photoprotection of a sunscreen against erythema. The SPF is defined as a ratio of the ultraviolet energy required to produce minimal erythema on protected skin to that required to produce the same minimal erythema on unprotected skin in the same individual. Amounts of the sunscreen used are preferably from about 2% to about 20%, and more preferably from about 4% to about 14%. Suitable sunscreens include, but are not limited to those approved for use in the United States, Japan, Europe and Australia. The compositions of the present invention preferably comprise an SPF of about 2 to about 30, preferably about 4 about 30, and more preferably about 4 to about 15.

In some embodiments, the compositions of the present invention comprise one or more UVA absorbing sunscreen actives that absorb UV radiation having a wavelength of from about 320 nm to about 400 nm. Suitable UVA absorbing sunscreen actives include, but are not limited to dibenzoylmethane (See e.g., Lowe and Shaath (eds.), Sunscreens: Development, Evaluation, and Regulatory Aspects, Marcel Dekker, Inc.) derivatives, anthranilate derivatives such as methylanthranilate and homomethyl, 1-N-acetylanthranilate, and mixtures thereof. The UVA absorbing sunscreen active is preferably present in an amount sufficient to provide broad spectrum UVA protection either independently, or in combination with, other UV protective actives which may be present in the composition.

Suitable UVA sunscreen actives include dibenzoylmethane sunscreen actives and their derivatives. They include, but are not limited to, those selected from 2-methyldibenzoylmethane, 4-methyldibenzoylmethane, 4-isopropyldibenzoylmethane, 4-tert-butyldibenzoylmethane, 2,4-dimethyldibenzoylmethane, 2,5-dimethyldibenzoylmethane, 4,4′-diisopropylbenzoylmethane, 4-(1,1-dimethylethyl)-4′-methoxydibenzoylmethane, 2-methyl-5-isopropyl-4′-methoxydibenzoylmethane, 2-methyl-5-tert-butyl-4′-methoxy-dibenzoylmethane, 2,4-dimethyl-4′-methoxydibenzoylmethane, 2,6-dimethyl-4′-tert-butyl-4′methoxydibenzoylmethane, and mixtures thereof. Preferred dibenzoyl sunscreen actives include those selected from 4-(1,1-dimethylethyl)-4′-methoxydibenzoylmethane, 4-isopropyldibenzoylmethane, and mixtures thereof. A preferred sunscreen active is 4-(1,1-dimethylethyl)-4′-methoxydibenzoylmethane.

The sunscreen active 4-(1,1-dimethylethyl)-4′-methoxydibenzoylmethane, which is also known as butyl methoxydibenzoylmethane or “avobenzone,” is commercially available under the names of Parsol® 1789 from Givaudan Roure (International) S. A., and Eusolex® 9020 from Merck & Co., Inc. The sunscreen 4-isoproplydibenzoylmethane, which is also known as isopropyldibenzoylmethane, is commercially available from Merck under the name of Eusolex® 8020.

In some embodiments, the compositions of the present invention further include one or more UVB sunscreen actives that absorb(s) UV radiation having a wavelength of about 290 nm to about 320 nm. The compositions comprise an amount of the UVB sunscreen active that is safe and effective in providing UVB protection either independently, or in combination with, other UV protective actives which may be present in the compositions. The compositions comprise from about 0.1% to about 20%, preferably from about 0.1% to about 12%, and more preferably from about 0.5% to about 8% by weight, of each UVB absorbing organic sunscreen, or as mandated by the relevant regulatory authority(s).

A variety of UVB sunscreen actives are suitable for use herein (See e.g., U.S. Pat. No. 5,087,372; U.S. Pat. No. 5,073,371; U.S. Pat. No. 5,073,372; U.S. Pat. No. 4,937,370; and U.S. Pat. No. 4,999,186). Preferred UVB sunscreen actives are selected from 2-ethylhexyl-2-cyano-3,2-ethylhexyl N,N-dimethyl-p-aminobenzoate, p-amino-benzoic acid, oxybenzone, homomethyl salicylate, octyl salicylate, 4,4′-methoxy-t-butyldibenzoylmethane, 4-isopropyl dibenzoylmethane, 3-benzylidene camphor, 3-(4-methylbenzylidene) camphor, 3-diphenylacrylate, 2-phenyl-benzimidazole-5-sulphonic acid (PBSA), cinnamate esters and their derivatives such as 2-ethylhexyl-p-methoxycinnamate, salicylate esters and their derivatives such as triethanolamine salicylate, ethylhexyl salicylate, octyldimethyl para-aminobenzoic acid, camphor derivatives and their derivatives, and mixtures thereof. Preferred organic sunscreen actives include 2-ethylhexyl-2-cyano-3,3-diphenylacrylate, 2-phenyl-benzimidazole-5-sulphonic acid (PBSA), octyl-p-methoxycinnamate, and mixtures thereof. Salt and acid neutralized forms of the acidic sunscreens are also useful herein.

Thus, in some embodiments, the present invention provides compositions comprising any organic UV-A and UV-B filter, for example but not limited to the following:

CAS-Nr. Nr. Compound (=Acid) 1 4-Aminobenzoicacid 150-13-0 2 3-(4′-Trimethylammonium)-benzylidenbornan-2-on- 52793-97-2 methylsulfate 3 3,3,5-Trimethyl-cyclohexyl-salicylate 118-56-9 (Homosalatum) 4 2-Hydroxy-4-methoxy-benzophenon 131-57-7 (Oxybenzonum) 5 2-Phenylbenzimidazol-5-sulfonic acid and their Calcium-, 27503-81-7 Sodium- and Triethanolaminosalts 6 3,3′-(1,4-Phenylendimethin)-bis(7,7-dimethyl- 90457-82-2 2-oxobicyclo[2.2.1]heptan-1-methansolfonicacid) and salts thereof 7 4-Bis(polyethoxy)amino-benzoesaurepolyethoxy-ethylester 113010-52-9 8 4-Dimethylamino-benzoicacid-2-ethylhexylester 21245-02-3 9 Salicylicacid-2-ethylhexylester 118-60-5 10 4-Methoxy-cinnamicacid-2-isoamylester 71617-10-2 11 4-Methoxy-cinnamicacid-2-ethylhexylester 5466-77-3 12 2-Hydroxy-4-methoxy-benzophenon-5-sulfonicacid- 4065-45-6 (Sulisobenzonum) and the sodiumsalt 13 3-(4′-Sulfobenzyliden)-bornan-2-on and salts thereof 58030-58-6 14 3-Benzylidenbornan-2-on 16087-24-8 15 1-(4′-Isopropylphenyl)-3-phenylpropan-1,3-dion 63260-25-9 16 4-Isopropylbenzylsalicylat 94134-93-7 17 3-Imidazol-4-yl-acrylicacid und ihr Ethylester 104-98-3 18 2-Cyano-3,3-diphenylacrylicacidethylester 5232-99-5 19 2-Cyano-3,3-diphenylacrylicacid-2′-ethylhexylester 6197-30-4 20 Menthyl-o-aminobenzoat oder: 134-09-8 5-Methyl-2-(1-methylethyl)-2-aminobenzoat 21 Glyceryl p-aminobenzoat oder: 136-44-7 4-Aminobenzoicacid-1-glyceryl-ester 22 2,2′-Dihydroxy-4-methoxybenzophenon (Dioxybenzone) 131-53-3 23 2-Hydroxy-4-methoxy-4-methylbenzophenon 1641-17-4 (Mexenon) 24 Triethanolamin Salicylat 2174-16-5 25 Dimethoxyphenylglyoxalsaure oder: 4732-70-1 3,4-dimethoxy-phenyl-glyoxal-saures Natrium 26 3-(4′Sulfobenzyliden)-bornan-2-on und seine Salze 56039-58-8 27 4-tert.-Butyl-4′-methoxy-dibenzoylmethan 70356-09-1 28 2,2′,4,4′-Tetrahydroxybenzophenon 131-55-5 29 2,2′-Methylen-bis-[6-(2H-benzotriazol-2-yl)-4-(1,1,3,3,- 103597-45-1 tetramethylbutyl)phenol] 30 2,2′-(1,4-Phenylen)-bis-1H-benzimidazol-4,6- 180898-37-7 disulfonicacid, Sodiumsalt 31 2,4-bis-[4-(2-Ethylhexyloxy)-2-hydroxy]phenyl- 187393-00-6 6-(4-methoxyphenyl)-(1,3,5)-triazin 32 3-(4-Methylbenzyliden)-campher 36861-47-9 33 4-Bis(polyethoxy)paraaminobenzoicacidpolyethoxyethylester 113010-52-9 34 2,4-Dihydroxybenzophenon 131-56-6 35 2,2′-Dihydroxy-4,4′-dimethoxybenzophenon-5,5′- 3121-60-6 disodiumsulfonat 36 Benzoicacid, 2-[4-(diethylamino)-2-hydroxybenzoyl]-, hexylester 302776-68-7 37 2-(2H-Benzotriazol-2-yl)-4-methyl-6-[2-methyl-3-[1,3,3,3- 155633-54-8 tetramethyl-1-[(trimethylsilyl)oxy]disiloxanyl]propyl]phenol 38 1,1-[(2,2′-Dimethylpropoxy)carbonyl]-4,4-diphenyl-1,3-butadien 363602-15-7

In some embodiments, at least one agent is added to any of the compositions useful in the present invention to stabilize the UVA sunscreen to prevent it from photo-degrading on exposure to UV radiation and thereby maintaining its UVA protection efficacy. A wide range of compounds are reported to have these stabilizing properties and should be chosen to complement both the UVA sunscreen and the composition as a whole (See e.g., U.S. Pat. Nos. 5,972,316; 5,968,485; 5,935,556; 5,827,508; and WO 00/06110). Preferred examples of stabilizing agents for use in the present invention include 2-ethylhexyl-2-cyano-3,3-diphenylacrylate, ethyl-2-cyano-3,3-diphenylacrylate, 2-ethylhexyl-3,3-diphenylacrylate, ethyl-3,3-bis(4-methoxyphenyl)acrylate, diethylhexyl 2,6 napthalate and mixtures thereof (Symrise Chemical Company).

In some preferred embodiments, the present invention provides cosmetic and/or topical dermatological preparations suitable for use as skin protection creams, cleansing milks, sun screen lotions, nourishing creams, day creams, night creams etc. In some embodiments, the present invention finds use a components of drug (i.e., pharmaceutical) compositions. In additional embodiments, the present invention finds use in decorative cosmetics (e.g., make-up formulations).

In some particularly preferred embodiments, the present invention provides sunscreens useful in cosmetic and/or skin care preparations. In addition to the active ingredient used according to the embodiments of the present invention, in some embodiments, these preparations preferably additionally comprise at least one broadband filter and/or at least one UVA filter substance and/or at least one UVB filter substance and/or at least one inorganic pigment.

In yet further embodiments, the present invention provides personal care compositions which have UV protection components, but which are not primarily sunscreens. For example, in some embodiments, UV-A and/or UV-B filter substances are incorporated into day creams and/or hair care compositions.

In additional embodiments, the personal care compositions of the present invention comprise cosmetically active ingredients, auxiliaries and/or additives, as are customarily used in such preparations (e.g., antioxidants, preservatives, bacteriocides, perfumes, antifoams, dyes, pigments which have a coloring action, thickeners, surface-active substances, emulsifiers, emollients, moisturizers and/or humectants, fats, oils, waxes or other customary constituents of a cosmetic or dermatological formulation, such as alcohols, polyols, polymers, foam stabilizers, electrolytes, organic solvents or silicone derivatives). Indeed it is contemplated that various compounds will find use in the various embodiments of the present invention, as appropriate for the product and the user.

In some embodiments, at least one agent is added to any of the compositions useful in the present invention to improve the skin substantivity of those compositions, particularly to enhance their resistance to being washed off by water or rubbed off. Examples include, but are not limited to, acrylates/C₁₂₋₂₂ alkylmethacrylate copolymer, acrylate/acrylate copolymer, dimethicone, dimethiconol, graft-copoly (dimethylsiloxane/1-butyl methacrylate), lauryl dimethicone, PVP/Hexadecane copolymer, PVP/Eicosene copolymer, tricontanyl PVP and trimethoxysiloxysilicate.

In addition to organic sunscreens, in some embodiments, the compositions of the present invention additionally comprise inorganic physical sunblocks (See e.g., TFA International Cosmetic Ingredient Dictionary, 6^(th) Edition, pp. 1026-28 and 1103 [1995]; Sayre et al., J. Soc. Cosmet. Chem., 41:103-109 [1990]; and Lowe et al., supra). Preferred inorganic physical sunblocks include zinc oxide and titanium dioxide and mixtures thereof.

When used in preferred embodiments, the physical sunblocks are present in an amount such that the present compositions are transparent on the skin (i.e., non-whitening), preferably from about 0.5% to about 20%, preferably from about 0.5% to about 10%, and more preferably from about 0.5% to 5% by weight. When titanium dioxide is used, it can have an anatase, rutile or amorphous structure. Manufacturers of micronized grade titanium dioxide and zinc oxide for sunscreen use include, but are not limited to Tayca Corporation, Uniqema, Shinetsu Chemical Corporation, Kerr-McGee, Nanophase, Nanosource, Sachtleben, Elementis, and BASF Corporation, as well as their distribution agents and those companies that further process the material for sunscreen use. Physical sunblock particles (e.g., titanium dioxide and zinc oxide) can be uncoated or coated with a variety of materials including but not limited to amino acids, aluminum compounds such as alumina, aluminum stearate, aluminum laurate, and the like; carboxylic acids and their salts (e.g., stearic acid and its salts); phospholipids, such as lecithin; organic silicone compounds; inorganic silicone compounds such as silica and silicates and mixtures thereof. In some preferred embodiments, the compositions of the present invention comprise from about 0.1% to about 15%, preferably from about 0.1% to about 7%, and more preferably from about 0.5% to about 5%, by weight, of inorganic sunscreen.

In addition to the deleterious effects of some emulsifiers, exposure to other factors is known to harm skin and hair. For example, the harmful effect of the ultraviolet portion of solar radiation on the skin is generally known. While rays having a wavelength of less than 290 nm (i.e., the UVC region) are absorbed by the ozone layer in the earth's atmosphere, rays in the range between 290 nm and 320 nm (i.e., the UVB region), cause erythema, simple sunburn or even burns of varying severity. The erythema activity maximum of sunlight is given as the relatively narrow region around 308 nm.

Numerous compounds are known to provide protection against harmful UVB radiation. Most commonly, these compounds are derivatives of 3-benzylidenecamphor, of 4-aminobenzoic acid, of cinnamic acid, of salicylic acid, of benzophenone, and of 2-phenyl-benzimidazole.

It is also important to have available filter substances for the range between about 320 nm and about 400 nm, the UVA region, since its rays can also cause damage. For a long time it was incorrectly assumed that the long-wave UV-A radiation having a wavelength of between 320 nm and 400 nm had only a negligible biological action and that, accordingly, the UV-B rays were responsible for most photodamage to the human skin. However, numerous recent studies have shown that UV-A radiation is much more harmful than UV-B radiation with regard to the triggering of photodynamic, specifically phototoxic, reactions and chronic changes in the skin. In addition, the harmful effects of UV-B radiation can be further intensified by exposure to UV-A radiation.

It has been shown that UV-A radiation by itself and under very normal everyday conditions, is sufficient to damage collagen and elastin fibers, which are of essential importance for the structure and strength of the skin, within a short period. This leads to chronic light-induced changes in the skin, such that the skin prematurely “ages.” The clinical appearance of skin aged by light typically includes increased wrinkles and lines, and an irregular, furrowed relief. In addition, the skin areas affected by light-induced skin aging often show irregular pigmentation. In some cases, brown patches, keratoses, carcinomas, or malignant melanomas arise. Skin prematurely aged as a result of everyday UV exposure is also characterized has having lowered activity of the Langerhans cells and slight, chronic inflammation.

Approximately 90% of the ultraviolet radiation which reaches the Earth consists of UV-A rays. While amount of UV-B radiation reaching Earth varies widely depending on numerous factors (e.g., time of year and day and/or degree of latitude), the UV-A radiation levels that reach Earth remain relatively constant on a daily basis, irrespective of the time of year and day or geographical factors. Additionally, the majority of UV-A radiation penetrates the living epidermis, while about 70% of the UV-B rays are retained by the horny layer. Preventive protection against UV-A rays, for example by applying light protection filter substances in the form of a cosmetic or dermatological formulation to the skin, is therefore of fundamental importance.

In general, the light absorption behavior of light protection filter substances is very well known and documented, largely due to the fact that most industrialized countries have positive lists for the use of such substances, which impose very strict standards on the documentation that accompanies each product which incorporates these substances. For the concentration of the substances in the finished formulations, the absorbance values provide a guide, since interaction with substances within the skin or the surface of the skin itself often presents variables that may impact how well the compositions perform on each individual. However, it is usually difficult to estimate beforehand, how uniformly and thickly the filter substance is distributed in and on the horny layer of the skin.

To test UV-A protection performance, use is usually made of the IPD method (IPD 5 immediate pigment darkening) known to those in the art. This method is similar to the determination of the sun protection factor, and provides a method which indicates how much longer skin protected with the light protection composition can be irradiated with UV-A radiation before the pigmentation which occurs is the same as that produced for unprotected skin.

Another test method which has become established throughout Europe is the Australian standard AS/NZS 2604:1997. In this method, the absorption of the preparation in the UV-A region is measured. In order to satisfy the standard, the preparation must absorb at least 90% of the UV-A radiation in the region 320-360 nm.

Of concern in the formulation of sunscreen compositions is that the use concentration of known light protection filter substances which also exhibit high filter action in the UV-A region are often limited by the very fact that they are combined with other substances which are in the form of solids. Thus, there are certain formulation difficulties associate with achieving relatively high sun protection factors and UV-A protection performance. However, those of skill in the art are generally aware of means to overcome and/or compensate for these difficulties.

As light protection filter substances are generally expensive and some light protection filter substances are additionally difficult to incorporate into cosmetic and/or dermatological preparations in relatively high concentrations, some embodiments of the present invention were designed to provide simple and cost-effective preparations which, despite having unusually low concentrations of conventional UV-A light protection filter substances, nevertheless achieve acceptable or even high UV-A protection performance.

However, as known in the art, UV radiation can also lead to photochemical reactions which produce products that interfere with the skin's metabolism. These photochemical reaction products are predominantly free-radical compounds (e.g., hydroxyl radicals). Undefined free-radical photoproducts which form in the skin itself can also exhibit uncontrolled secondary reactions as a result of their high reactivity. However, singlet oxygen, a non-free-radical excited state of the oxygen molecule, can also arise during UV irradiation, as can short-lived epoxides and many others. Singlet oxygen, for example, differs from normal triplet oxygen (free-radical ground state) by virtue of its increased reactivity. However, excited, reactive “free-radical” triplet states of the oxygen molecule also exist. Thus, in order to prevent these reactions, antioxidants and/or free-radical scavengers find use in cosmetic and/or dermatological formulations.

The compounds which are commonly used as light protection agents in cosmetic and/or dermatological light protection formulations are generally characterized as providing good light protection. However, they have the disadvantage that it is sometimes difficult to incorporate them into the desired formulations in a satisfactory manner.

As indicated above, the sun protection factor (SPF) indicates how much longer the skin protected with the light protection composition can be irradiated before the erythema reaction which occurs is the same as for unprotected skin (i.e., ten times as long compared with unprotected skin for an SPF=10). Consumers are very aware of the meaning of “SPF” and choose skin and/or hair care products based on the SPF values indicated on products. Consumers expect to receive reliable information from manufacturers regarding the sun protection factor, largely due to increased public awareness of the association between excess sun exposure and skin cancer, as well as premature aging. In addition, in some parts of the world, the degradation of the ozone layer is a major concern. Depending upon the skin type and the sun exposure expected, consumers choose products with a lower or a higher SPF. However, there appears to be a tendency for consumers to select relatively high SPF factors, particularly for products to be applied to children and those with fair skin. In some embodiments, the present invention provides compositions with relatively low concentrations of conventional light protection filter substances, yet with SPF values that are acceptable to consumers.

In some preferred embodiments, the basic constituents of the sunscreen preparations provided by the present invention include: water or aqueous solutions; aqueous ethanolic solutions; natural oils and/or chemically modified natural oils and/or synthetic oils; fats, waxes and other natural and synthetic fatty substances, preferably esters of fatty acids with alcohols of low carbon number (e.g., with isopropanol, propylene glycol or glycerol), or esters of fatty alcohols with alkanoic acids of low carbon number or with fatty acids; alcohols, diols or polyols of low carbon number, and ethers thereof, preferably ethanol, isopropanol, propylene glycol, glycerol, ethylene glycol, ethylene glycol monoethyl or monobutyl ether, propylene glycol monomethyl, monoethyl or monobutyl ether, diethylene glycol monomethyl or monoethyl ether and analogous products. In alternatively preferred embodiments, mixtures of two or more of these constituents find use in the present invention.

In some preferred embodiments, the composition of the present invention also includes preservatives. Such preservatives include, but are not limited to pentylene glycol, ethylene diamine tetra acetate (EDTA) and their salts, chlorhexidine (and its diacetate, dihydrochloride, digluconate derivatives), 1,1,1-trichloro-2-methyl-2-propanol, parachloro metaxylenol, polyhexamethylenebiguanide hydrochloride, dehydroacetic acid, diazolidinyl urea, 2,4-dichlorobenzyl alcohol, 4,4-dimethyl-1,3-oxazolidine, formaldehyde (e.g., 37% aqueous solution, with 10-15% methanol to avoid polymerization), glutaraldehyde, dimethylidantoin, imidazolidinyl urea, 5-Chloro-2-methyl-4-isothiazolin-3-one, ortho-phenylphenol, 4-hydroxybenzoic acid esters (e.g., “paraben”) and its methyl-, ethyl-, propyl-, isopropyl-, butyl-, and isobutyl-esters, trichlosan, 2-phenoxyethanol, phenyl mercuric acetate, borate, nitrate, quaternium-15, salicylate, salicylic acid and its salts, calcium, calcium sorbate, sorbic acid and its salts, iodopropanyl butylcarbamate zinc pyrithione, benzyl alcohol, 5-bromo-5-nitro-1,3-dioxane, 2-bromo-2-nitropropane-1,3-diol, benzoic acid and its salts, sulfites, bisulfites, phenyoxyethanol, chloroxylenol, diazolidinyl urea, methylparabens, propylparabens, isoproplyparabens, isobutylparabens, butylparabens, ethylparaben, phenoxyethanol PG, and benzalkonium chloride.

In still further embodiments, preservatives, such as those used in food and feed applications find use in various compositions of the present invention. The following table provides a list of such compounds, as well as the E number for each compound. However, it is not intended that the present invention be limited to these specific preservatives, as it is contemplated that additional preservatives will find use in various embodiments of the present invention.

Examples of Food Grade Preservatives That Find Use in Embodiments of the Present Invention 200 Sorbic acid 201 Sodium sorbate 202 Potassium sorbate 203 Calcium sorbate 210 Benzoic acid 211 Sodium benzoate 212 Potassium benzoate 213 Calcium benzoate 214 Ethyl p-hydroxybenzoate 215 p-hydroxybenzoic ethyl ester Na salt 216 n-propyl p-hydroxybenzoate 217 p-hydroxybenzoic-n-propyl ester Na salt 218 methyl p-hydroxybenzoate 219 p-hydroxybenzoic methyl ester Na salt 220 Sulfur dioxide 221 Sodium sulfite 222 Sodium hydrogensulfite 223 Sodium disulfite 224 Potassium disulfite 226 Calcium sulfite 227 Calcium hydrogen sulfite 228 Potassium hydrogen sulfite 230 Biphenyl (Diphenyl) 231 Orthophenylphenol 232 Sodium orthophenylphenoxide 233 Thiabendazole 235 Natamycin 236 Formic acid 237 Sodium formate 238 Calcium formate 239 Hexamethylenetetramine 249 Potassium nitrite 250 Sodium nitrite 251 Sodium nitrate 252 Potassium nitrate 280 Propionic acid 281 Sodium propionate 282 Calcium propionate 283 Potassium propionate 290 Carbon dioxide

Additional preservatives that find use in various embodiments include but are not limited to dibromodicyanobutane (2-bromo-2-bromomethylglutarodinitrile), 3-iodo-2-propinylbutylcarbamate, 2-bromo-2-nitropropane-1,3-diol, imidazolidinylurea, 5-chloro-2-methyl-4-isothiazolin-3-one, 2-chloroacetamide, benzalkonium chloride, benzyl alcohol, and formaldehyde donors. Further preservatives that find use in various embodiments of the present invention include phenyl hydroxyalkyl ethers, in particular the compounds known as “phenoxyethanol,” due to their bactericidal and fungicidal effects on a number of microorganisms.

A variety of optional ingredients such as neutralizing agents, perfumes and perfume solubilizing agents, and coloring agents, also find use in some of the compositions herein. It is preferred that any additional ingredients enhance the skin softness/smoothness benefits of the product. In addition it is preferred that any such ingredients do not negatively impact the aesthetic properties of the product.

Other optional materials include keratolytic agents, as well as water-soluble and/or solubilizable preservatives preferably at a level of from about 0.1% to about 5% (e.g., Germall 115, methyl, ethyl, propyl and butyl esters of hydroxybenzoic acid, benzyl alcohol, DMDM hydantoin iodopropanyl butylcarbanate available under the trade name Glydant Plus from Lonza; EDTA, EUXYL® K400, Bromopol (2-bromo-2-nitropropane-1,3-diol) and phenoxypropanol); anti-bacterials (e.g., IRGASAN®) and phenoxyethanol (preferably at levels of from about 0.1% to about 5%); as well as soluble or colloidally-soluble moisturizing agents such as hyaluronic acid, chondroitin sulfate, and starch-grafted sodium polyacrylates (e.g., SANWET® IM-1000, IM-1500 and IM-2500, available from Celanese Superabsorbent Materials, Portsmith, Va., See e.g., U.S. Pat. No. 4,076,663; vitamins such as vitamin A, vitamin C, vitamin E and derivatives thereof and building blocks thereof such as phytantriol, and vitamin K and components thereof such as the fatty alcohol dodecatrienol; alpha and beta hydroxyacids; aloe vera; sphingosines and phytosphingosines, cholesterol; skin whitening agents; N-acetyl cysteine; colouring agents; antibacterial agents such as TCC/TCS, also known as triclosan and trichlorocarbon; perfumes and perfume solubilizers. Examples of alpha hydroxy acids include glycolic acid, lactic acid, malic acid, citric acid, glycolic acid in conjunction with ammonium glycolate, alpha-hydroxy ethanoic acid, alpha-hydroxyoctanoic acid, alpha-hydroxycaprylic acid, hydroxycaprylic acid, mixed fruit acid, tri-alpha hydroxy fruit acids, triple fruit acid, sugar cane extract, alpha hydroxy and botanicals, I-alpha hydroxy acid and glycomer in crosslinked fatty acids (e.g., alpha nutrium). Preferred examples of alpha hydroxy acids are glycolic acid and lactic acid. It is preferred that alpha hydroxy acids are used in levels of up to about 10%. It is not intended that the present invention be limited to any particular compound derived from any particular source, as any suitable additive compound, whether obtained from natural sources or through synthesis in the laboratory find use in the present invention.

Other optional materials include water-soluble or solubilizable preservatives preferably at a level of from about 0.1% to about 5% each, such as Germall 115, methyl, ethyl, propyl and butyl esters of hydroxybenzoic acid, benzyl alcohol, DMDM hydantoin iodopropanyl butylcarbanate available under the trade name Glydant Plus from Lonza, EDTA, Euxyl® K400, Bromopol (2-bromo-2-nitropropane-1,3-diol), pentylene glycol and phenoxypropanol; anti-bacterials such as Irgasan® and phenoxyethanol (preferably at levels of from 0.1% to about 5%). Antibacterial agents such as TCC/TCS, also known as triclosan and trichlorocarbon are also useful in compositions of the present invention.

Yet other antimicrobial agents are likewise suitable for use in various embodiments of the present invention, including but not limited to 2,4,4′-trichloro-2′-hydroxydiphenyl ether (i.e., IRGASAN®), 1,6-di(4-chlorophenylbiguanido)hexane (i.e., CHLORHEXIDIN), 3,4,4′-trichlorocarbanilide, quaternary ammonium compounds, oil of cloves, mint oil, thyme oil, triethyl citrate, FARNESOL® (3,7,11-trimethyl-2,6,10-dodecatrien-1-ol) and the active ingredients and/or active ingredient combinations described in DE-37 40 186, DE-39 38 140, DE-42 04 321, DE-42 29 707, DE-43 09 372, DE-44 11 664, DE-1 95 41 967, DE-1 95 43 695, DE-1 95 43 696, DE-1 95 47 160, DE-1 96 02 108, DE-196 02 110, DE-196 02 111, DE-196 31 003, DE-196 31 004, DE-196 34 019, DE-42 29 737, DE-42 37 081, DE-43 24 219, DE-44 29 467, DE-44 23 410, and DE-195 16 705, all of which are hereby incorporated by reference. In still further embodiments, sodium hydrogencarbonate is also included in some compositions of the present invention. However, it is not intended that the present invention be limited to any particular antimicrobial nor combination of anti-microbial, as various compounds having such effects will find use in various embodiments of the present invention.

In additional embodiments of the personal care compositions of the present invention, compounds such as anti-irritants and/or anti-inflammatory actives are included. In some embodiments, batyl alcohol (a-octadecyl glyceryl ether), selachyl alcohol (a-9-octadecenyl glyceryl ether), chimyl alcohol (a-hexadecyl glyceryl ether), bisabolol, and/or panthenol are included. However, it is not intended that the present invention be limited to the incorporation of any specific anti-irritant(s) and/or anti-inflammatory(ies), as various compounds suitable for such applications find use in the present invention.

Neutralizing agents suitable for use in neutralizing acidic group containing hydrophilic gelling agents herein include sodium hydroxide, potassium hydroxide, ammonium hydroxide, monoethanolamine, diethanolamine, amino methyl propanol, tris-buffer and triethanolamine.

Other optional materials that find use in the present invention include any of the numerous functional and/or active ingredients known to those skilled in the art (See e.g., McCutcheon's Functional Materials, North American and International Editions, MC Publishing Co. [2003]) As indicated above, non-limiting examples include keratolytic agents; soluble or colloidally-soluble moisturizing agents such as hyaluronic acid and chondroitin sulfate; vitamins such as vitamin A, vitamin C, vitamin E, vitamin K and derivatives thereof and building blocks thereof; phytantriol; fatty alcohols such as dodecatrienol; alpha and beta hydroxyacids; aloe vera; sphingosines and phytosphingosines, cholesterol; skin whitening agents; N-acetyl cysteine; coloring agents; Examples of alpha hydroxy acids include glycolic acid, lactic acid, malic acid, and citric acid (whether derived synthetically or from natural sources and whether used alone or in combination) and their esters or relevant buffered combinations. Other examples of alpha-hydroxy acids include: alpha-hydroxy ethanoic acid, alpha-hydroxyoctanoic acid, alpha-hydroxycaprylic acid, and hydroxycaprylic acid. Preferred examples of alpha hydroxy acids include glycolic acid and lactic acid. It is preferred that alpha hydroxy acids are used in levels of up to about 10%.

Optional materials include pigments that, where water-insoluble, contribute to and are included in the total level of oil phase ingredients. Pigments suitable for use in the compositions of the present invention can be organic and/or inorganic. Also included within the term “pigment” are materials having a low color or luster, such as matte finishing agents, light scattering agents, and formulation aids such as micas, seracites, and carbonate salts. Further examples of suitable pigments include titanium dioxide, iron oxides, glutamate iron oxides, zinc oxide, bismuth oxychloride, ultramarine blue (all of which may be either pre-dispersed and/or pre-coated or not) D&C dyes and lakes, FD&C colors, natural color additives such as carmine, and mixtures thereof. Depending upon the type of composition, a mixture of pigments is usually used in preferred embodiments of the present invention. Preferred pigments for use herein from the viewpoint of moisturization, skin feel, skin appearance and emulsion compatibility are treated pigments. In some embodiments, the pigments are treated with compounds, including but not limited to amino acids, silicones, lecithin and ester oils.

In some embodiments, the present invention provides compositions comprising pigments, including, but not limited to inorganic pigments based on metaloxides and/or other in water slightly soluble or insoluble metal compounds such as zinc oxides (ZnO), titanium (TiO₂), iron (e.g., Fe₂O₃), zirconium (ZrO₂), silica (SiO₂), manganese (e.g., MnO), aluminium (Al₂O₃), cer (e.g., Ce₂O₃), and mixed compositions of these oxides, as well as blends thereof. In some preferred embodiments, the metaloxides are microfine, while in alternative preferred embodiments, the metaloxides are pigment grade. In yet additional embodiments, the pigments are “coated” such that they are surface treated. In some preferred embodiments, the coating comprises a thin, hydrophobic film layer, while in other embodiments, the coating comprises a thin, hydrophilic film layer.

As used herein, the terms “pigment,” “color pigment,” and “dye” used in reference to the compositions of the present invention encompasses any compound that provides a color to the composition and/or imparts a color to the surface (e.g., skin and/or hair) to which the composition is applied. In some embodiments, the dyes and pigments are chosen from the list of cosmetic colorants provided by the Cosmetics Directive or the EC. In most cases, these dyes and pigments are identical to the dyes approved for foods. Preferred pigments/dyes include for example, titanium dioxide, mica, iron oxides (e.g., Fe₂O₃, Fe₃O₄, FeO(OH)) and/or tin oxide. Advantageous pigments/dyes include for example, carmine, Berlin blue, chrome oxide green, ultramarine blue and/or manganese violet. In some preferred embodiments, the pigments/dyes include, but are not limited to those in the following table. The Colour Index Numbers (CIN) those known in the art (See, Society of Dyers and Colourists, Rowe Colour Index, 3rd Edition, Bradford, England, [1971]).

CHEMICAL OR OTHER NAME CIN COLOR Pigment Green 10006 green Acid Green 1 10020 green 2,4-Dinitrohydroxynaphthalene-7-sulfonic acid 10316 yellow Pigment Yellow 1 11680 yellow Pigment Yellow 3 11710 yellow Pigment Orange 1 11725 orange 2,4-Dihydroxyazobenzene 11920 orange Solvent Red 3 12010 red 1-(2′-Chloro-4′-nitro-1′-phenylazo)-2-hydroxynaphthalene 12085 red Pigment Red 3 12120 red Ceres red; Sudan red; Fat Red G 12150 red Pigment Red 112 12370 red Pigment Red 7 12420 red Pigment Brown 1 12480 brown 4-(2′-Methoxy-5′-sulfodiethylamido-1′-phenylazo)-3-hydroxy- 12490 red 5″-chloro-2″,4″-dimethoxy-2-naphthanilide Disperse Yellow 16 12700 yellow 1-(4-Sulfo-1-phenylazo)-4-aminobenzene-5-sulfonic acid 13015 yellow 2,4-Dihydroxyazobenzene-4′-sulfonic acid 14270 orange 2-(2,4-Dimethylphenylazo-5-sulfo)-1-hydroxynaphthalene-4-sulfonic acid 14700 red 2-(4-Sulfo-1-naphthylazo)-1-naphthol-4-sulfonic acid 14720 red 2-(6-Sulfo-2,4-xylylazo)-1-naphthol-5-sulfonic acid 14815 red 1-(4′-Sulfophenylazo)-2-hydroxynaphthalene 15510 orange 1-(2-Sulfo-4-chloro-5-carboxy-1-phenylazo)-2- 15525 red hydroxynaphthalene 1-(3-Methylphenylazo-4-sulfo)-2-hydroxynaphthalene 15580 red 1-(4′,(8′)-Sulfonaphthylazo)-2-hydroxynaphthalene 15620 red 2-Hydroxy-1,2′-azonaphthalene-1′-sulfonic acid 15630 red 3-Hydroxy-4-phenylazo-2-naphthylcarboxylic acid 15800 red 1-(2-Sulfo-4-methyl-1-phenylazo)-2-naphthyl-carboxylic acid 15850 red 1-(2-Sulfo-4-methyl-5-chloro-1-phenylazo)-2- 15865 red hydroxynaphthalene-3-carboxylic acid 1-(2-Sulfo-1-naphthylazo)-2-hydroxynaphthalene-3-carboxylic acid 15880 red 1-(3-Sulfo-1-phenylazo)-2-naphthol-6-sulfonic acid 15980 orange 1-(4-Sulfo-1-phenylazo)-2-naphthol-6-sulfonic acid 15985 yellow Allura Red 16035 red 1-(4-Sulfo-1-naphthylazo)-2-naphthol-3,6-disulfonic acid 16185 red Acid Orange 10 16230 orange 1-(4-Sulfo-1-naphthylazo)-2-naphthol-6,8-disulfonic acid 16255 red 1-(4-Sulfo-1-naphthylazo)-2-naphthol-3,6,8-trisulfonic acid 16290 red 8-Amino-2-phenylazo-1-naphthol-3,6-disulfonic acid 17200 red Acid Red 1 18050 red Acid Red 155 18130 red Acid Yellow 121 18690 yellow Acid Red 180 18736 red Acid Yellow 11 18820 yellow Acid Yellow 17 18965 yellow 4-(4-Sulfo-1-phenylazo)-1-(4-sulfophenyl)-5-hydroxy- 19140 yellow pyrazolone-3-carboxylic acid Pigment Yellow 16 20040 yellow 2,6-(4′-Sulfo-2″,4″-dimethyl)bisphenylazo)-1,3- 20170 orange dihydroxybenzene Acid Black 1 20470 black Pigment Yellow 13 21100 yellow Pigment Yellow 83 21108 yellow Solvent Yellow 21230 yellow Acid Red 163 24790 red Acid Red 73 27290 red 2-[4′-(4″-Sulfo-1″-phenylazo)-7′-sulfo-1′-naphthylazo]-1- 27755 black hydroxy-7-aminonaphthalene-3,6-di-sulfonic acid 4′-[(4″-Sulfo-1″-phenylazo)-7′-sulfo-1′-naphthylazo]-1- 28440 black hydroxy-8-acetylaminonaphthalene-3,5-disulfonic acid Direct Orange 34, 39, 44, 46, 60 40215 orange Food Yellow 40800 orange trans-β-Apo-8′-carotinaldehyde (C30) 40820 orange trans-Apo-8′-carotinic acid (C30)-ethyl ester 40825 orange Canthaxanthin 40850 orange Acid Blue 1 42045 blue 2,4-Disulfo-5-hydroxy-4′-4″-bis(diethylamino)triphenylcarbinol 42051 blue 4-[(4-N-Ethyl-p-sulfobenzylamino)phenyl(4-hydroxy-2- 42053 green sulfophenyl)(methylene)-1-(N-ethyl-N-p-sulfobenzyl)-2,5- cyclohexadienimine] Acid Blue 7 42080 blue (N-Ethyl-p-sulfobenzylamino)phenyl(2- 42090 blue sulfophenyl)methylene-(N-ethyl-N-p-sulfobenzyl)D2,5- cyclohexadienimine Acid Green 9 42100 green Diethyldisulfobenzyldi-4-amino-2-chloro-di-2-methyl- 42170 green fuchsonimmonium Basic Violet 14 42510 violet Basic Violet 2 42520 violet 2′-Methyl-4′-(N-ethyl-N-m-sulfobenzyl)amino-4″-(N- 42735 blue diethyl)amino-2-methyl-N-ethyl-N-m- sulfobenzylfuchsonimmonium 4′-(N-Dimethyl)amino-4″-(N-phenyl)aminonaphtho-N- 44045 blue dimethyl-fuchsonimmonium 2-Hydroxy-3,6-disulfo-4,4′-bisdimethylaminonaphtho- 44090 green fuchsonimmonium Acid Red 52 45100 red 3-(2′-Methylphenylamino)-6-(2′-methyl-4′-sulfophenylamino)- 45190 violet 9-(2″-carboxyphenyl)xanthenium salt Acid Red 50 45220 red Phenyl-2-oxyfluorone-2-carboxylic acid 45350 yellow 4,5-Dibromofluorescein 45370 orange 2,4,5,7-Tetrabromofluorescein 45380 red Solvent Dye 45396 orange Acid Red 98 45405 red 3′,4′, 5′, 6′-Tetrachloro-2,4,5,7-tetrabromofluorescein 45410 red 4,5-Diiodofluorescein 45425 red 2,4,5,7-Tetraiodofluorescein 45430 red Quinophthalone 47000 yellow Quinophthalonedisulfonic acid 47005 yellow Acid Violet 50 50325 violet Acid Black 2 50420 black Pigment Violet 23 51319 violet 1,2-Dioxyanthraquinone, calcium-aluminum complex 58000 red 3-Oxypyrene-5,8,10-sulfonic acid 59040 green 1-Hydroxy-4-N-phenylaminoanthraquinone 60724 violet 1-Hydroxy-4-(4′-methylphenylamino)anthraquinone 60725 violet Acid Violet 23 60730 violet 1,4-Di(4′-methylphenylamino)anthraquinone 61565 green 1,4-Bis(o-sulfo-p-toluidino)anthraquinone 61570 green Acid Blue 80 61585 blue Acid Blue 62 62045 blue N,N′-Dihydro-1,2,1′,2′-anthraquinone azine 69800 blue Vat Blue 6; Pigment Blue 64 69825 blue Vat Orange 7 71105 orange Indigo 73000 blue Indigo-disulfonic acid 73015 blue 4,4′-Dimethyl-6,6′-dichlorothioindigo 73360 red 5,5′-Dichloro-7,7′-dimethylthioindigo 73385 violet Quinacridone Violet 19 73900 violet Pigment Red 122 73915 red Pigment Blue 16 74100 blue Phthalocyanine 74160 blue Direct Blue 86 74180 blue Chlorinated Phthalocyanines 74260 green Natural Yellow 6, 19; Natural Red 1 75100 yellow Bixin, Nor-Bixin 75120 orange Lycopene 75125 yellow trans-alpha-, beta- and gamma-carotene 75130 orange Keto- and/or hydroxyl derivates of carotene 75135 yellow Guanine or pearlizing agent 75170 white 1,7-Bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione 75300 yellow Complex salt (Na, Al, Ca) of carminic acid 75470 red Chlorophyll a and b; copper compounds of chlorophylls and 75810 green Chlorophyllins Aluminum 77000 white Hydrated alumina 77002 white Hydrous aluminum silicates 77004 white Ultramarine 77007 blue Pigment Red 101 und 102 77015 red Barium sulfate 77120 white Bismuth oxychloride and its mixtures with mica 77163 white Calcium carbonate 77220 white Calcium sulfate 77231 white Carbon 77266 black Pigment Black 9 77267 black Carbo medicinalis vegetabilis 77268 black Chromium oxide 77288 green Chromium oxide, hydrous 77289 green Pigment Blue 28, Pigment Green 14 77346 green Pigment Metal 2 77400 brown Gold 77480 brown Iron oxides and hydroxides 77489 orange Iron oxide 77491 red Iron oxide, hydrated 77492 yellow Iron oxide 77499 black Mixtures of iron (II) and iron(III)hexacyano-ferrate 77510 blue Pigment White 18 77713 white Manganese ammonium diphosphate 77742 violet Manganese phosphate; Mn3(PO4)2 □ 7 H20 77745 red Silver 77820 white Titanium dioxide and its mixtures with mica 77891 white Zinc oxide 77947 white 6,7-Dimethyl-9-(1′-D-ribityl)-isoalloxazine, lactoflavine yellow Sugar coloring brown Capsanthin, capsorubin orange Betanin red Benzopyrylium salts, Anthocyans red Aluminum, zinc, magnesium and calcium stearate white Bromothymol blue blue Bromocresol green green Acid Red 195 red

In yet further embodiments, compositions of the present invention further comprise one or more substances from the following group: 2,4-dihydroxyazobenzene, 1-(2′-chloro-4′-nitro-1′-phenylazo)-2-hydroxynaphthalene, Ceres Red, 2-(4-sulfo-1-naphthylazo)-1-naphthol-4-sulfonic acid, calcium salt of 2-hydroxy-1,2′-azonaphthalene-1′-sulfonic acid, calcium and barium salts of 1-(2-sulfo-4-methyl-1-phenylazo)-2-naphthylcarboxylic acid, calcium salt of 1-(2-sulfo-1-naphthylazo)-2-hydroxynaphthalene-3-carboxylic acid, aluminum salt of 1-(4-sulfo-1-phenylazo)-2-naphthol-6-sulfonic acid, aluminum salt of 1-(4-sulfo-1-naphthylazo)-2-naphthol-3,6-disulfonic acid, 1-(4-sulfo-1-naphthylazo)-2-naphthol-6,8-disulfonic acid, aluminum salt of 4-(4-sulfo-1-phenylazo)-1-(4-sulfophenyl)-5-hydroxypyrazolone-3-carboxylic acid, aluminum and zirconium salts of 4,5-dibromofluorescein, aluminum and zirconium salts of 2,4,5,7-tetrabromofluorescein, 3′,4′,5′,6′-tetrachloro-2,4,5,7-tetrabromofluorescein and its aluminum salt, aluminum salt of 2,4,5,7-tetraiodofluorescein, aluminum salt of quinophthalone disulfonic acid, aluminum salt of indigo disulfonic acid, red and black iron oxide (CIN: 77 491 (red) and 77 499 (black)), iron oxide hydrate (CIN: 77 492), manganese ammonium diphosphate and titanium dioxide.

In yet further embodiments, oil-soluble natural dyes, such as, for example, paprika extracts, β-carotene or cochenille find use in the present invention.

In yet additional embodiments, gel cream compositions of the present invention comprise pearlescent pigments. In some preferred embodiments, various pearlescent pigments find use in the present invention, including but not limited to “natural pearlescent pigments” (e.g., “pearl essence” [guanine/hypoxanthine mixed crystals from fish scales], “mother of pearl” [ground mussel shells]), and “monocrystalline pearlescent pigments” (e.g., bismuth oxychloride [BiOCl]); and “layer substrate pigments” (e.g. mica/metal oxide).

Bases for pearlescent pigments include, but are not limited to pulverulent pigments, castor oil dispersions of bismuth oxychloride and/or titanium dioxide, bismuth oxychloride and/or titanium dioxide on mica. The luster pigment listed under CIN 77163, for example, is particularly advantageous.

An additional group of pearlescent pigments based on mica/metal oxide find particular use in the present invention is provided below.

COATING/LAYER GROUP THICKNESS COLOR Silver-white pearlescent pigments TiO2: 40-60 nm silver Interference pigments TiO2: 60-80 nm yellow TiO2: 80-100 nm red TiO2: 100-140 nm blue TiO2: 120-160 nm green Color luster pigments Fe2O3 bronze Fe2O3 copper Fe2O3 red Fe2O3 red-violet Fe2O3 red-green Fe2O3 black Combination pigments TiO2/Fe2O3 gold shades TiO2/Cr2O3 green TiO2/Berlin blue deep blue TiO2/carmine red

In some preferred embodiments, the pearlescent pigments available from Merck under the trade names Timiron, Colorona or Dichrona find use in the present invention. However, it is not intended that the present invention be limited to the specific pigments listed herein. Indeed, pearlescent pigments that find use in the present invention are obtainable from numerous sources. For example, other substrates apart from mica can be coated with further metal oxides, such as, for example, silica and the like. SiO₂ particles coated with, for example, TiO₂ and Fe₂O₃ (“ronaspheres”), which are sold by Merck and are particularly suitable for the optical reduction of fine lines find use in the present invention.

In alternative embodiments, the substrate (e.g., mica) is not included. In some preferred embodiments, particular preference is given to pearlescent pigments prepared using SiO₂. Such pigments, which may also additionally have goniochromatic effects, are available, for example, under the trade name Sicopearl Fantastico, available from BASF.

In additional embodiments, pigments obtained from Engelhard/Mearl based on calcium sodium borosilicate which have been coated with titanium dioxide also find use. These are available under the name Reflecks. In addition to the color, as a result of their particle size of from 40 nm to 180 mm, they have a glitter effect.

In yet further embodiments, effect pigments which are available under the trade name Metasomes Standard/Glitter in various colors (yellow, red, green, blue) from Flora Tech find use in the compositions of the present invention. The glitter particles are present here in the mixtures with various auxiliaries and dyes (such as, for example, the dyes with the Colour Index (CI) Numbers 19140, 77007, 77289, 77491).

In some embodiments, the dyes and pigments are present either individually or in a mixture. In alternative embodiments, they are mutually coated with one another, different coating thicknesses generally giving rise to different color effects. In some embodiments, the total amount of dyes and color-imparting pigments is chosen from a range of concentrations (e.g., from about 0.1% by weight to about 30% by weight; preferably from about 0.5 to about 15% by weight; and most preferably from about 1.0 to about 10% by weight, in each case based on the total weight of the preparations).

In preferred embodiments, the pH of the compositions herein is in the range from about 3.5 to about 10, preferably from about 4 to about 8, and more preferably from about 5 to about 7, wherein the pH of the final composition is adjusted by addition of acidic, basic or buffer salts as necessary, depending upon the composition of the forms and the pH-requirements of the compounds.

The compositions of the present invention are prepared by standard techniques well known to those skilled in the art. In general the aqueous phase and/or the oil phase are prepared separately, with materials of similar phase partitioning being added in any order. If the final product is an emulsion, the two phases are then combined with vigorous stirring and/or homogenization as necessary, to reduce the size of the internal phase droplets. Any ingredients in the formulation with high volatility, or which are susceptible to hydrolysis or decomposition at high temperatures, are added with gentle stirring towards the end of the process, post emulsification if applicable. Dosage frequency and amount will depend upon the desired performance criteria.

In some embodiments of the present invention, method of decreasing VEGF activity are provided. In these embodiments, the methods comprise applying to an organism in need thereof an effective amount of any one of the compounds set forth herein. In additional preferred embodiments, the present invention provides compounds for treatment of an organism in need thereof, including humans and other animals.

In some still further embodiments, the present invention comprises at least one creatine and/or creatine derivative. Creatine has the following structure:

In some preferred embodiments of the personal care compositions of the present invention creatine phosphate, creatine sulfate, creatine acetate, creatine ascorbate, and/or derivatives esterified at the carboxyl group with mono- or polyfunctional alcohols find use.

In some additional embodiments, the personal care compositions of the present invention contain L-carnitine [3-hydroxy-4-(trimethylammonio)butyrobetaine]. Acylcarnitines have the following general structure:

where R is chosen from the group of branched and unbranched alkyl radicals having up to 10 carbon atoms, and find use in some embodiments of the present invention. In some preferred embodiments, propionylcarnitine and/or acetylcarnitine find use. Both enantiomers (D and L form), as well as mixtures and racemates of the D- and L-forms find use in some personal care compositions of the present invention.

In some further embodiments, the active ingredients of the present invention include, but are not limited to sericoside, pyridoxol, vitamin K, biotin, and aroma substances. In addition, it is not intended that the active ingredients present in the personal care compositions of the present invention be limited to any particular constituent and/or mixture(s) of actives. Indeed, it is intended that various actives and mixtures of actives will find use in various embodiments of the present invention. It is also not intended that the concentration(s) of such actives be limited to any particular level. In some embodiments, the concentration is from about 0.001 to about 30% by weight, while in other embodiments it is from about 0.05 to about 20% by weight, and in still further embodiments, it is from about 0.1 to about 10% by weight, based on the total weight of the preparation. It is further contemplated that those of skill in the art will formulate personal care compositions of the present invention with active(s) concentrations that are suitable for the intended use of the compositions.

The yet further embodiments, the present invention provides methods for the preparation of the compositions of the present invention. In some embodiments, these methods include combining and heating the constituents of the oil phase and/or the water phase separately, and then combining them together with stirring. In some preferred embodiments, the phases are homogenized. In some particularly preferred embodiments, the compositions are stirred with moderate to high input of energy, advantageously using a gear rim dispersing machine at a rotary number up to at most 10000 rpm (preferably in the range from about 2500 to about 7700 rpm).

6.2 EXPERIMENTAL

The present invention is described in further detail in the following Examples which are not in any way intended to limit the scope of the invention as claimed. The attached Figures are meant to be considered as integral parts of the specification and description of the invention. All references cited are herein specifically incorporated by reference for all that is described therein. The following examples are offered to illustrate, but not to limit the claimed invention

In the experimental disclosure which follows, the following abbreviations apply PI (proteinase inhibitor), BBI (Bowman-Birk Inhibitor from Glycine max Acc. No. P01055), BBI-AV (Bowman-Birk Inhibitor Anti-VegF), STI (Soybean Trypsin inhibitor from Glycine max); VEGF and VegF (vascular endothelial growth factor); BBdb (Bowman Birk Inhibitor from Dolichos biflorus Acc. No. AAK97765), BBsb3 (Bowman Birk Inhibitor from Glycine max (soybean) protease inhibitor IV or D-II), and BBtc (Bowman Birk Inhibitor from Torresea cearensis), FGF-5 (fibroblast growth factor 5), TGFβ (Transforming growth factor β), TNFα (Tumor necrosis factor α), ppm (parts per million); M (molar); mM (millimolar); μM (micromolar); nM (nanomolar); mol (moles); mmol (millimoles); μmol (micromoles); nmol (nanomoles); μm (grams); mg (milligrams); μg (micrograms); pg (picograms); L (liters); ml and mL (milliliters); μl and μL (microliters); cm (centimeters); mm (millimeters); μm (micrometers); nm (nanometers); U (units); V (volts); MW (molecular weight); sec (seconds); min(s) (minute/minutes); h(s) and hr(s) (hour/hours); ° C. (degrees Centigrade); QS (quantity sufficient); ND (not done); NA (not applicable); rpm (revolutions per minute); H₂O (water); dH₂O (deionized water); (HCl (hydrochloric acid); aa (amino acid); bp (base pair); kb (kilobase pair); kD (kilodaltons); cDNA (copy or complimentary DNA); DNA (deoxyribonucleic acid); ssDNA (single stranded DNA); dsDNA (double stranded DNA); dNTP (deoxyribonucleotide triphosphate); RNA (ribonucleic acid); MgCl₂ (magnesium chloride); NaCl (sodium chloride); w/v (weight to volume); v/v (volume to volume); g (gravity); OD (optical density); Dulbecco's phosphate buffered solution (DPBS); SOC (2% Bacto-Tryptone, 0.5% Bacto Yeast Extract, 10 mM NaCl, 2.5 mM KCl); Terrific Broth (TB; 12 g/l Bacto Tryptone, 24 g/l glycerol, 2.31 g/l KH₂PO₄, and 12.54 g/l K₂HPO₄); OD₂₈₀ (optical density at 280 nm); OD₆₀₀ (optical density at 600 nm); A₄₀₅ (absorbance at 405 nm); Vmax (the maximum initial velocity of an enzyme catalyzed reaction); PAGE (polyacrylamide gel electrophoresis); PBS (phosphate buffered saline [150 mM NaCl, 10 mM sodium phosphate buffer, pH 7.2]); PBST (PBS+0.25% TWEEN® 20); PEG (polyethylene glycol); PCR (polymerase chain reaction); RT-PCR (reverse transcription PCR); SDS (sodium dodecyl sulfate); Tris (tris(hydroxymethyl)aminomethane); HEPES (N-[2-Hydroxyethyl]piperazine-N-[2-ethanesulfonic acid]); HBS (HEPES buffered saline); SDS (sodium dodecylsulfate); bME, BME and βME (beta-mercaptoethanol or 2-mercaptoethanol); Tris-HCl (tris[Hydroxymethyl]aminomethane-hydrochloride); Tricine (N-[tris-(hydroxymethyl)-methyl]-glycine); CHES (2-(N-cyclo-hexylamino) ethane-sulfonic acid); TAPS (3-{[tris-(hydroxymethyl)-methyl]-amino}-propanesulfonic acid); CAPS (3-(cyclo-hexylamino)-propane-sulfonic acid; DMSO (dimethyl sulfoxide); DTT (1,4-dithio-DL-threitol); Glut and GSH (reduced glutathione); GSSG (oxidized glutathione); TCEP (Tris[2-carboxyethyl]phosphine); Ci (Curies) mCi (milliCuries); μCi (microCuries); TLC (thin layer achromatography); Ts (tosyl); Bn (benzyl); Ph (phenyl); Ms (mesyl); Et (ethyl), Me (methyl); Taq (Thermus aquaticus DNA polymerase); Klenow (DNA polymerase I large (Klenow) fragment); rpm (revolutions per minute); EGTA (ethylene glycol-bis(β-aminoethyl ether) N,N,N′,N′-tetraacetic acid); EDTA (ethylenediaminetetracetic acid); bla (β-lactamase or ampicillin-resistance gene); GE Healthcare (GE Healthcare, Chalfont St. Giles, United Kingdom); DNA2.0 (DNA2.0, Menlo Park, Calif.); OXOID (Oxoid, Basingstoke, Hampshire, UK); Megazyme (Megazyme International Ireland Ltd., Bray Business Park, Bray, Co., Wicklow, Ireland); Corning (Corning Life Sciences, Corning, N.Y.); (NEN (NEN Life Science Products, Boston, Mass.); Pharma AS (Pharma AS, Oslo, Norway); Dynal (Dynal, Oslo, Norway); Bio-Synthesis (Bio-Synthesis, Lewisville, Tex.); ATCC (American Type Culture Collection, Rockville, Md.); Gibco/BRL (Gibco/BRL, Grand Island, N.Y.); Sigma (Sigma Chemical Co., St. Louis, Mo.); Pharmacia (Pharmacia Biotech, Pisacataway, N.J.); NCBI (National Center for Biotechnology Information); Applied Biosystems (Applied Biosystems, Foster City, Calif.); Clontech (CLONTECH Laboratories, Palo Alto, Calif.); Operon Technologies (Operon Technologies, Inc., Alameda, Calif.); MWG Biotech (MWG Biotech, High Point, N.C.); Oligos Etc (Oligos Etc. Inc, Wilsonville, Oreg.); Bachem (Bachem Bioscience, Inc., King of Prussia, Pa.); Difco (Difco Laboratories, Detroit, Mich.); Mediatech (Mediatech, Herndon, Va.; Santa Cruz (Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.); BioVeris (BioVeris Corp., Gaithersburg, Md.); Oxoid (Oxoid Inc., Ogdensburg, N.Y.); Worthington (Worthington Biochemical Corp., Freehold, N.J.); GIBCO BRL or Gibco BRL (Life Technologies, Inc., Gaithersburg, Md.); Millipore (Millipore, Billerica, Mass.); Bio-Rad (Bio-Rad, Hercules, Calif.); Invitrogen (Invitrogen Corp., San Diego, Calif.); NEB (New England Biolabs, Beverly, Mass.); Sigma (Sigma Chemical Co., St. Louis, Mo.); Pierce (Pierce Biotechnology, Rockford, Ill.); Takara (Takara Bio Inc. Otsu, Japan); Roche (Hoffmann-La Roche, Basel, Switzerland); EM Science (EM Science, Gibbstown, N.J.); Qiagen (Qiagen, Inc., Valencia, Calif.); Biodesign (Biodesign Intl., Saco, Me.); Aptagen (Aptagen, Inc., Herndon, Va.); Molecular Devices (Molecular Devices, Corp., Sunnyvale, Calif.); R&D Systems (R&D Systems, Minneapolis, Minn.); Stratagene (Stratagene Cloning Systems, La Jolla, Calif.); Marsh (Marsh Biosciences, Rochester, N.Y.); Bio-Tek (Bio-Tek Instruments, Winooski, Vt.); (Biacore (Biacore, Inc., Piscataway, N.J.); PeproTech (PeproTech, Rocky Hill, N.J.); SynPep (SynPep, Dublin, Calif.); and Microsoft (Microsoft, Inc., Redmond, Wash.).

Example 1 Production of BCE103-BBI Fusion Proteins in B. subtilis

In this Example, experiments conducted to produce BCE103-BBI fusion proteins in B. subtilis are described. The DNA sequence of the synthetic gene (Operon Technologies) coding for the pro-BBI protein with a C-terminal hexa-histidine tag used in these experiments is:

(SEQ ID NO:10) AACCTGCGTCTGTCTAAGCTTGGCCTGCTTATGAAATCAGACCATCAGCA CAGCAATGACGATGAGAGCTCTAAACCCTGTTGCGATCAATGCGCATGTA CGAAATCAAATCCTCCACAGTGTCGGTGTTCCGATATGCGTCTGAATAGC TGTCATAGTGCATGCAAAAGCTGTATCTGCGCCCTGAGTTATCCAGCTCA ATGTTTTTGCGTCGACATCACGGACTTCTGCTATGAGCCATGTAAACCAA GCGAGGACGATAAAGAGAACCATCATCACCATCACCAT

The protein sequence of pro-BBI with a C-terminal hexa-histidine tagged coded for by the above synthetic gene is:

(SEQ ID NO:11) NLRLSKLGLLMKSDHQHSNDDESSKPCCDQCACTKSNPPQCRCSDMRLNS CHSACKSCICALSYPAQCFCVDITDFCYEPCKPSEDDKENHHHHHH

The portion of the DNA sequence of the synthetic gene that codes for the major mature form of BBI is:

(SEQ ID NO:12) GACGATGAGAGCTCTAAACCCTGTTGCGATCAATGCGCATGTACGAAATC AAATCCTCCACAGTGTCGGTGTTCCGATATGCGTCTGAATAGCTGTCATA GTGCATGCAAAAGCTGTATCTGCGCCCTGAGTTATCCAGCTCAATGTTTT TGCGTCGACATCACGGACTTCTGCTATGAGCCATGTAAACCAAGCGAGGA CGATAAAGAGAAC

The protein sequence of the major mature form of BBI coded by the above synthetic gene is:

(SEQ ID NO:13) DDESSKPCCDQCACTKSNPPQCRCSDMRLNSCHSACKSCICALSYPAQCF CVDITDFCYEPCKPSEDDKEN

The PCR primers used to amplify the BBI gene for fusion to the BCE103 cellulase expression cassette in the pJ103 vector were:

BBIfusion_FW: (SEQ ID NO:14) 5′ CAGCACGGATCCAGACGATGAGAGCTCTAAACCC 3′ BBIHindIII_RV: (SEQ ID NO:15) 5′ CTGCAGAAGCTTAAAAATAAAAAAACGGATTTCCTTCAGGAAATCCG TCCTCTGTTAACTTTTAGTTCTCTTTATCGTCCTCGC 3′ BBIHIS-HindIII_RV: (SEQ ID NO:16) 5′ CTGCAGAAGCTTAAAAATAAAAAAACGGATTTCCTTCAGGAAATCCG TCCTCTGTTAACTTTTAATGGTGATGGTGATGATGGTTCTC 3′

The sequence of the aprE-BCE103-BBI-HisTag expression cassette (EcoRI-HindIII) that was cloned into the pJM103 integration vector is provided in FIG. 1. A schematic plasmid map of the pJM103BBIHis expression vector is provided in FIG. 2.

The alkaline cellulase (BCE103) gene (See, van Soligen, U.S. Pat. No. 6,063,611, hereby incorporated by reference) fused to the B. subtilis aprE promoter and signal sequence, was cloned from pUCAPR103 (Shaw et al., J. Mol. Biol., 320:303-309 [2002]) as an EcoRI-BamHI fragment (i.e., a fragment that carries the coding sequence of the BCE103 catalytic domain and first cellulose binding domain linker only) into pJM103 (Perego, “Integrational vectors for genetic manipulation in Bacillus subtilis” In, Bacillus subtilis and Other Gram-positive Bacteria: Biochemistry, Physiology, and Molecular Genetics, Sonenshein, Hoch, and Losick (eds), American Society for Microbiology, Washington D.C., pp. 615-624 [1993]). A gene encoding the soybean Bowman-Birk protease inhibitor (BBI) (Swiss-Prot Accession #P01055; See, Odani and Ikenaka, J. Biochem., 71: 839-848 [1972]) with a C-terminal hexa-histidine tag (His-Tag) was synthesized by Operon Technologies (See, DNA sequence above). The BBI gene was amplified by PCR with primers (all primers were synthesized by MWG Biotech, Oligos Etc., or Operon Technologies) that generated a 5′ BamHI site in the correct reading frame with the BCE103 gene, and at the 3′ end introduced a strong transcriptional terminator (LAT, from the Bacillus licheniformis α-amylase gene) after the end of the BBI gene with a 3′ HindIII site for cloning into the pJM103 vector.

PCR fragments with or without a C-terminal His-Tag were generated with the primers BBIfusion_FW (SEQ ID NO:14) and BBIHISHindIII_RV (SEQ ID NO:16), or BBIfusion_FW (SEQ ID NO:14) and BBI-HindIII_RV (SEQ ID NO:15), respectively, using the synthetic BBI gene as a template. Unless indicated otherwise, PCR reactions were typically performed on a thermocycler for 30 cycles with High Fidelity Platinum Taq polymerase (Invitrogen) according to the instructions of the supplier (with an annealing temperature of 55° C.). The PCR fragments were cloned as BamHI-HindIII fragments into pJM103 carrying the aprE-BCE103 expression cassette. The correct gene sequence was verified by DNA sequencing.

Thus, as shown in FIG. 1, the N-terminus of the mature coding region of the BBI gene (with or without the His-Tag) was fused in frame to the C-terminal coding region of the first CBD (cellulose binding domain) linker sequence coded by the BCE103 cellulase gene. Thereby, the two CBD's of BCE103 (Shaw et al., supra) are replaced by BBI in the final expression vectors, pJM103BBI or pJM103BBIhis (See, FIG. 2). The aprE promoter controls the expression of the BCE103-BBI gene fusions (See, Ferrari et al., J. Bact., 170:289-295 [1988]; and Henner et al., J. Bact., 170: 296-300 [1988]).

Competent Bacillus subtilis cells, BG3934comK (degU^(Hy)32, oppA, ΔspoIIE3501, ΔaprE, ΔnprE, Δepr, ΔispA, Δbpr, amyE::xylRPxylAcomK-phleo), were transformed with the expression plasmids, pJM103BBI or pJM103BBIhis. The bacteria were made competent by the induction of the comK gene under control of a xylose inducible promoter (Hahn et al., Mol. Microbiol., 21:763-775 [1996]). The transformants were selected on Luria Broth agar (LA) plates containing 5 μg/ml chloramphenicol. To increase the expression by gene amplification, colonies were streaked and grown several times on LA plates with 25 μg/ml chloramphenicol until the growth rate with the antibiotic was similar to growth rate in the absence of chloramphenicol. The BCE103-BBI fusion protein was produced by growth in shake flasks at 37° C. in TSB medium (Tryptone Soya Broth from OXOID, 30 g/L) or in MBD medium, a MOPS based defined medium. MBD medium was made essentially as described (Neidhardt et al., J. Bacteriol., 119: 736-747 [1974]), except NH₄Cl₂, FeSO₄, and CaCl₂ were left out of the base medium, 3 mM K₂HPO₄ was used, and the base medium was supplemented with 60 mM urea, 75 g/L glucose, and 1% soytone. Also, the micronutrients were made up as a 100× stock containing in one liter, 400 mg FeSO₄.7H₂O, 100 mg MnSO₄.H₂O, 100 mg ZnSO₄.7H₂O, 50 mg CuCl₂.2H₂O, 100 mg CoCl₂.6H₂O, 100 mg NaMoO₄.2H₂O, 100 mg Na₂B₄O₇.10H₂O, 10 ml of 1M CaCl₂, and 10 ml of 0.5 M sodium citrate.

BCE103-BBI fusion protein could be easily visualized in samples from cell free supernatants (after 24 h of growth in TSB medium or 48 h in MBD medium) as the major protein band on SDS-PAGE gels (10% NuPAGE in MES buffer, run as described by the manufacturer, Invitrogen) running at ˜44 kDa by using standard protein stains (e.g. GelCode Blue Stain Reagent; Pierce). The identity of the BCE103-BBI fusion protein was verified by immunoblots of SDS-PAGE gels using the protocols supplied by the manufacturer (BM Chromogenic Western Blotting Kit; Roche Applied Science using an anti-HisTag antibody or an anti-BCE103 cellulase polyclonal antibody for detection).

To determine the BCE103 activity, cellulase degradation was assessed qualitatively on LA cellulase indicator plates (with 1% carboxymethylcellulose stained with 0.2% Congo Red, or with 0.5% azo-CM-cellulose, Megazyme), or quantitatively by a direct assay in Assay Buffer (100 mM Tris pH 8.6, 0.005% Tween-80) on the culture broth using a the synthetic substrate, 4-nitrophenyl □-D-cellobioside (Sigma), using methods known in the art (See e.g., van Tilbeurgh et al., Meth. Enzymol., 160:45-59 [1988]).

Trypsin inhibitory assays were performed in Assay Buffer to determine the BBI activity. Specifically, a standard curve was generated by making eleven 1:1 serial dilutions (100 μL BBI+100 μL Assay Buffer) of a 2 pg/mL standard BBI solution. The BBI standard was purified from a 1 mg/ml Trypsin-Chymotrypsin Inhibitor (Sigma Cat. #T-9777) solution in 20 mM MES pH 6.0 using a hydrophobic interaction column (POROS HP2, Phenyl column, Applied Biosystems). The column was equilibrated with 20 mM MES pH 6.0, loaded with 5 mg of the inhibitor, washed with the equilibration buffer, and then the BBI was eluted with water. Unknown BBI samples to be tested in the inhibitory assay were diluted as necessary, so that two or more data points would fall within the standard curve (usually 1:10, 1:100, 1:200, 1:1000, 1:2000 sample dilutions were tested and then the dilutions fine tuned if necessary). Each diluted BBI standard or sample, 20 μL, was added to 80 μL of 50 ng/ml bovine pancreatic trypsin (Worthington) (made by diluting a stock 1 mg/mL trypsin solution into Assay Buffer). For convenience, the standards and samples were arrayed in 96 well microtiter plates. The reactions were mixed and incubated 15 min at 25° C. After the incubation, 100 μL of the 0.5 mg/ml trypsin substrate (diluted in Assay Buffer from a 100 mg/ml solution in DMSO), Suc-AAPR-pNA (succinyl-Ala-Ala-Pro-Arg-para-nitroanilide, Bachem), was added, mixed and the OD (A₄₀₅) was monitored for 15 min, with 1 time point recorded every 12 sec using a Spectra Max 250 (Molecular Devices). The data points were used to determine the Vmax for each reaction. The standard curve was generated by plotting Vmax versus BBI concentration and was fitted to a four-parameter curve. All data fitting was done using software supplied by the manufacturer (Molecular Devices). The BBI concentration of the unknown samples was calculated from the standard curve. Alternatively, the BBI activity was measured using the same protocol but by determining bovine pancreatic chymotrypsin (Worthington) inhibition (chymotrypsin was used at the same concentration as trypsin and chymotrypsin activity was measured by adding 100 μL of a 0.4 mg/ml chymotrypsin substrate, succinyl-Ala-Ala-Pro-Phe-para-nitroanilide, Bachem).

Titers from shake flask runs (500 ml MBD medium in 2.8 L Fernbach 6 baffled flasks, 37° C., 225 rpm, harvested 60 h after of growth) typically ranged from 0.4-0.9 mg/ml BCE activity and 40-150 μg/ml BBI trypsin inhibitory activity. However, it is contemplated that titers likely could be improved further by optimizing the bacterial strain, culture medium and growth conditions (aeration, temperature, time of harvest, etc.).

In addition to the BCE103 fusion to wild-type BBI, fusion proteins to BBI variants and fusion proteins with various linkers between BCE103 and BBI were produced using the methods outlined above, as described in the following Examples. In addition, fusion proteins were also produced when the BBI was fused to the 2^(nd) CBD linker (BCE-cbdD-BBI; See, Example 4) making it possible to use the 1^(st) CBD to aid in the purification process.

Example 2 Production of Peptides Substituted into the BBI Reactive Site Loops as BCE103-BBI Fusion Proteins

In this Example, experiments conducted to produce peptides substituted into the BBI reactive site loops as BCE103-BBI fusion proteins are described. The primers, as well as other sequences used in the various steps of these experiments are provided below. The sequence of 12BBIck81 from the BCE103 fusion site (at the BamHI) to the end of the gene is provided in FIG. 3. The CK37281 peptide sequences (ACYNLYGWTC (SEQ ID NO:9) are inserted into both the trypsin and chymotrypsin inhibitory loops.

The primers used to introduce an EcoRI site in the BBI gene using QuikChange® site-directed mutagenesis (Stratagene) were:

BowBeco-F 5′-GATATGCGTCTGAATTCCTGTCATAGTGCAT (SEQ ID NO:17) BowBeco-R 5′-ATGCACTATGACAGGAATTCAGACGCATATC (SEQ ID NO:18)

The sequences of the DNA oligonucleotides that were annealed and cloned in the BBI gene (SacI-EcoRI) to replace the trypsin inhibitory loop with the VegF binding peptide CK37281 were:

1BBck81+ (SEQ ID NO:19) 5′-CTAAACCCTGTTGCGATCAATGCGCATGTTATAATTTGTATGGGTGG ACTTGTCGCTGCAGCGATATGCGTCTG 1BBck81− (SEQ ID NO:20) 5′-AATTCAGACGCATATCGCTGCAGCGACAAGTCCACCCATACAAATTA TAACATGCGCATTGATCGCAACAGGGTTTAGAGCT

The sequences of the DNA oligonucleotides that were annealed and cloned in the BBI gene (EcoRI-SalI) to replace the chymotrypsin inhibitory loop with the VegF binding peptide CK37281 were:

2BBck81+ (SEQ ID NO:21) 5′-AATTCCTGTCATAGTGCCTGCAAAAGCTGCGCATGTTATAACCTGTA CGGGTGGACCTGTTTTTGCG 2BBck81− (SEQ ID NO:22) 5′-TCGACGCAAAAACAGGTCCACCCGTACAGGTTATAACATGCGCAGCT TTTGCAGGCACTATGACAGG

The DNA sequences of the oligonucleotide pairs used to make cassettes to introduce peptides into the trypsin (SacI and EcoRI restriction sites) or chymotypsin (EcoRI and SalI restriction sites) reactive site loops of the synthetic BBI gene are provided below. These peptide coding sequences were then moved into the p2JM103BBI expression vector as SacI-SalI fragments.

Comstatin (1^(st) loop) (SEQ ID NO:23) CTAAACCCTGTTGCGATCAATGCGCATGTGTTGTTCAGGACTGGGGTCAC CACCGTTGTCGCTGCAGCGATATGCGTCTG and (SEQ ID NO:24) AATTCAGACGCATATCGCTGCAGCGACAACGGTGGTGACCCCAGTCCTGA ACAACACATGCGCATTGATCGCAACAGGGTTTAGAGCT Comstatin (2^(nd) loop) (SEQ ID NO:25) CAAAAGCTGTATCTGCGTTGTTCAGGACTGGGGTCACCACCGTTGTTTTT GCG and (SEQ ID NO:26) TCGACGCAAAAACAACGGTGGTGACCCCAGTCCTGAACAACGCAGATACA GCTTTTGCATG C2c (1^(st) loop) (SEQ ID NO:27) CTAAACCCTGTTGCGATCAATGCAGCTGTGGTCGTAAAATCCCGATCCAG TGTCGCTGCAGCGATATGCGTCTG and (SEQ ID NO:28) AATTCAGACGCATATCGCTGCAGCGACACTGGATCGGGATTTTACGACCA CAGCTGCATTGATCGCAACAGGGTTTAGAGCT C3c (1^(st) loop) (SEQ ID NO:29) CTAAACCCTGTTGCGATCAATGCGGTTGTGCTCGTTCTAACCTGGACGAA TGTCGCTGCAGCGATATGCGTCTG and (SEQ ID NO:30) AATTCAGACGCATATCGCTGCAGCGACATTCGTCCAGGTTAGAACGAGCA CAACCGCATTGATCGCAACAGGGTTTAGAGCT C4c (1^(st) loop) (SEQ ID NO:31) CTAAACCCTGTTGCGATCAATGCGGTTGTCAGCGTGCTCTGCCGATCCTG TGTCGCTGCAGCGATATGCGTCTG and (SEQ ID NO:32) AATTCAGACGCATATCGCTGCAGCGACACAGGATCGGCAGAGCACGCTGA CAACCGCATTGATCGCAACAGGGTTTAGAGCT C5c (1^(st) loop) (SEQ ID NO:33) CTAAACCCTGTTGCGATCAATGCCAGTGTGGTCGTCTGCACATGAAAACC TGTCGCTGCAGCGATATGCGTCTG and (SEQ ID NO:34) AATTCAGACGCATATCGCTGCAGCGACAGGTTTTCATGTGCAGACGACCA CACTGGCATTGATCGCAACAGGGTTTAGAGCT Xa1 (2^(nd) loop) (SEQ ID NO:35) AATTCCTGTCATAGTGCCTGCAAAAGCTGTATCTGCGCCCGTAGTTTGCC AGCTCAATGTTTTTGCG and (SEQ ID NO:36) TCGACGCAAAAACATTGAGCTGGCAAACTACGGGCGCAGATACAGCTTTT GCAGGCACTATGACAGG hSCC1 (1^(st) loop) (SEQ ID NO:37) CTAAACCCTGTTGCGATCAATGCAACTGTACGTACTCAACCCCTCCACAG TGTCGCTGCAGCGATATGCGTCTG and (SEQ ID NO:38) AATTCAGACGCATATCGCTGCAGCGACACTGTGGAGGGGTTGAGTACGTA CAGTTGCATTGATCGCAACAGGGTTTAGAGCT

The DNA sequences of oligonucleotide primer pairs used to introduce peptide sequences into the trypsin or chymotrypsin reactive site loops using a QuikChange® II XL site-directed mutagenesis kit (Stratagene) are provided below. The reactions were performed as outlined by the manufacturer and described in this Example. Twenty cycles were performed with extensions of 6 minutes at 68° C., denaturations of 50 s at 95° C., and annealings at 55° C. for 50 s. After the cycles, a final extension was performed at 68° C. for 20 minutes.

1A (2^(nd) loop) (SEQ ID NO:39) CTGTATCTGCAAACGCTCAAAATCTCGTGGCTGTTTTTGCGTCGACATCA C and (SEQ ID NO:40) CGCAAAAACAGCCACGAGATTTTGAGCGTTTGCAGATACAGCTTTTGCAT G 2B (2^(nd) loop) (SEQ ID NO:41) CTGTATCTGCTGGTATAATCAAATGACAACATGTTTTTGCGTCGACATCA C and (SEQ ID NO:42) CGCAAAAACATGTTGTCATTTGATTATACCAGCAGATACAGCTTTTGCAT G 4A (2^(nd) loop) (SEQ ID NO:43) CTGTATCTGCCATCAACTTGGCCCGAATTCATGTTTTTGCGTCGACATCA C and (SEQ ID NO:44) CGCAAAAACATGAATTCGGGCCAAGTTGATGGCAGATACAGCTTTTGCAT G 5A (2^(nd) loop) (SEQ ID NO:45) CTGTATCTGCCATCCGTGGGCACCGTATTCTTGTTTTTGCGTCGACATCA C and (SEQ ID NO:46) CGCAAAAACAAGAATACGGTGCCCACGGATGGCAGATACAGCTTTTGCAT G 6-1A (2^(nd) loop) (SEQ ID NO:47) CTGTATCTGCAATCTTCATTATCTTCAACAGTGTTTTTGCGTCGACATCA C and (SEQ ID NO:48) CGCAAAAACACTGTTGAAGATAATGAAGATTGCAGATACAGCTTTTGCAT G 7A (2^(nd) loop) (SEQ ID NO:49) CTGTATCTGCACACCGTCTCTTTATCGCCCGTGTTTTTGCGTCGACATCA C and (SEQ ID NO:50) CGCAAAAACACGGGCGATAAAGAGACGGTGTGCAGATACAGCTTTTGCAT G 8B (2^(nd) loop) (SEQ ID NO:51) CTGTATCTGCCTTACAGATCAATCTAAACCGTGTTTTTGCGTCGACATCA C and (SEQ ID NO:52) CGCAAAAACACGGTTTAGATTGATCTGTAAGGCAGATACAGCTTTTGCAT G 9A (2^(nd) loop) (SEQ ID NO:53) CTGTATCTGCGTTACAACATCAATGGGCATGTGTTTTTGCGTCGACATCA C and (SEQ ID NO:54) CGCAAAAACACATGCCCATTGATGTTGTAACGCAGATACAGCTTTTGCAT G 10B (2^(nd) loop) (SEQ ID NO:55) CTGTATCTGCCGCGCATCACCGTATGATTGGTGTTTTTGCGTCGACATCA C and (SEQ ID NO:56) CGCAAAAACACCAATCATACGGTGATGCGCGGCAGATACAGCTTTTGCAT G 11-1A (2^(nd) loop) (SEQ ID NO:57) CTGTATCTGCTCAACACAAAAAATTCCGCAATGTTTTTGCGTCGACATCA C and (SEQ ID NO:58) CGCAAAAACATTGCGGAATTTTTTGTGTTGAGCAGATACAGCTTTTGCAT G 12B (2^(nd) loop) (SEQ ID NO:59) CTGTATCTGCACACAATTTCGCTCTGCAACATGTTTTTGCGTCGACATCA C and (SEQ ID NO:60) CGCAAAAACATGTTGCAGAGCGAAATTGTGTGCAGATACAGCTTTTGCAT G 13A (2^(nd) loop) (SEQ ID NO:61) CTGTATCTGCCCGGATCATGTTCCGCATCTTTGTTTTTGCGTCGACATCA C and (SEQ ID NO:62) CGCAAAAACAAAGATGCGGAACATGATCCGGGCAGATACAGCTTTTGCAT G 15-1A (2^(nd) loop) (SEQ ID NO:63) CTGTATCTGCTCAGGCTTTCCGCTTTCTACATGTTTTTGCGTCGACATCA C and (SEQ ID NO:64) CGCAAAAACATGTAGAAAGCGGAAAGCCTGAGCAGATACAGCTTTTGCAT G 1A6 (1^(st) loop) (SEQ ID NO:65) TCAATGCGCATGTGAAGAGATCTGGACTATGCTTTGCCGGTGTTCCGATA TGCGTC and (SEQ ID NO:66) CGGAACACCGGCAAAGCATAGTCCAGATCTCTTCACATGCGCATTGATCG CAACAG 1A6 (2^(nd) loop) (SEQ ID NO:67) CAAAAGCTGTGCTTGTGAAGAGATCTGGACTATGCTTTGCTTTTGCGTCG ACATCACGG and (SEQ ID NO:68) ACGCAAAAGCAAAGCATAGTCCAGATCTCTTCACAAGCACAGCTTTTGCA TGCACTATG 1C2 (1^(st) loop) (SEQ ID NO:69) TCAATGCGCATGTTGGGCCCTTACTGTCAAAACATGCCGGTGTTCCGATA TGCGTC and (SEQ ID NO:70) CGGAACACCGGCATGTTTTGACAGTAAGGGCCCAACATGCGCATTGATCG CAACAGG 1C2 (2^(nd) loop) (SEQ ID NO:71) CAAAAGCTGTGCTTGTTGGGCCCTTACTGTCAAAACATGCTTTTGCGTCG ACATCACGG and (SEQ ID NO:72) ACGCAAAAGCATGTTTTGACAGTAAGGGCCCAACAAGCACAGCTTTTGCA TGCACTATG 2E2 (1^(st) loop) (SEQ ID NO:73) TCAATGCGCATGTCTTACAGTACTGTGGACTACATGCCGGTGTTCCGATA TGCGTC and (SEQ ID NO:74) CGGAACACCGGCATGTAGTCCACAGTACTGTAAGACATGCGCATTGATCG CAACAGG 2E2 (2^(nd) loop) (SEQ ID NO:75) CAAAAGCTGTGCTTGTCTTACAGTACTGTGGACTACATGCTTTTGCGTCG ACATCACGG and (SEQ ID NO:76) ACGCAAAAGCATGTAGTCCACAGTACTGTAAGACAAGCACAGCTTTTGCA TGCACTATG 2E5 (1^(st) loop) (SEQ ID NO:77) TCAATGCGCATGTACTCTTTGGAACAGATCTCCTTGCCGGTGTTCCGATA TGCGTC and (SEQ ID NO:78) CGGAACACCGGCAAGGAGATCTGTTCCAAAGAGTACATGCGCATTGATCG CAACAGG 2E5 (2^(nd) loop) (SEQ ID NO:79) CAAAAGCTGTGCTTGTACTCTTTGGAATCGATCTCCTTGCTTTTGCGTCG ACATCACGG and (SEQ ID NO:80) ACGCAAAAGCAAGGAGATCGATTCCAAAGAGTACAAGCACAGCTTTTGCA TGCACTATG FGFns (1^(st) loop) (SEQ ID NO:81) TCAATGCGCATGTACAAACATCGATTCTACTCCTTGCCGGTGTTCCGATA TGCGTC and (SEQ ID NO:82) CGGAACACCGGCAAGGAGTAGAATCGATGTTTGTACATGCGCATTGATCG CAACAGG FGFns (2^(nd) loop) (SEQ ID NO:83) CAAAAGCTGTGCTTGCACAAACATCGATTCTACTCCTTGTTTTTGCGTCG ACATCACGG and (SEQ ID NO:84) ACGCAAAAACAAGGAGTAGAATCGATGTTTGTGCAAGCACAGCTTTTGCA TGCACTATG FGFkr (1^(st) loop) (SEQ ID NO:85) TCAATGCGCATGTACAAAAATCGATCGTACTCCTTGCCGGTGTTCCGATA TGCGTC and (SEQ ID NO:86) CGGAACACCGGCAAGGAGTACGATCGATTTTTGTACATGCGCATTGATCG CAACAGG FGFkr (2^(nd) loop) (SEQ ID NO:87) CAAAAGCTGTGCTTGCACAAAAATCGATCGTACTCCTTGTTTTTGCGTCG ACATCACGG and (SEQ ID NO:88) ACGCAAAAACAAGGAGTACGATCGATTTTTGTGCAAGCACAGCTTTTGCA TGCACTATG FGFhI (1^(st) loop) (SEQ ID NO:89) TCAATGCGCATGTCACCTGCAGACAACTGAAACATGCCGGTGTTCCGATA TGCGTC and (SEQ ID NO:90) CGGAACACCGGCATGTTTCAGTTGTCTGCAGGTGACATGCGCATTGATCG CAACAGG FGFhI (2^(nd) loop) (SEQ ID NO:91) CAAAAGCTGTGCTTGCCACCTGCAGACAACTGAAACATGTTTTTGCGTCG ACATCACGG and (SEQ ID NO:92) ACGCAAAAACATGTTTCAGTTGTCTGCAGGTGGCAAGCACAGCTTTTGCA TGCACTATG FGFgy (1^(st) loop) (SEQ ID NO:93) TCAATGCGCATGTGGCTACTTCATCCCATCGATTTGCCGGTGTTCCGATA TGCGTC and (SEQ ID NO:94) CGGAACACCGGCAAATCGATGGGATGAAGTAGCCACATGCGCATTGATCG CAACAGG FGFgy (2^(nd) loop) (SEQ ID NO:95) CAAAAGCTGTGCTTGCGGCTACTTCATCCCATCGATTTGTTTTTGCGTCG ACATCACGG and (SEQ ID NO:96) ACGCAAAAACAAATCGATGGGATGAAGTAGCCGCAAGCACAGCTTTTGCA TGCACTATG MM005 (1^(st) loop) (SEQ ID NO:97) TCAATGCGCATGTTTACGTATCCTTGCTAACAAATGCCGGTGTTCCGATA TGCGTC and (SEQ ID NO:98) CGGAACACCGGCATTTGTTAGCAAGGATACGTAAACATGCGCATTGATCG CAACAGG MM005 (2^(nd) loop) (SEQ ID NO:99) CAAAAGCTGTGCTTGCTTACGTATCCTTGCTAACAAATGTTTTTGCGTCG ACATCACGG and (SEQ ID NO:100) ACGCAAAAACATTTGTTAGCAAGGATACGTAAGCAAGCACAGCTTTTGCA TGCACTATG MM007 (1^(st) loop) (SEQ ID NO:101) GCGATCAATGCGCCTGCAGAACTCAACCATATCCTTTATGTCGGTGTTCC GATATGCGTC and (SEQ ID NO:102) GGAACACCGACATAAAGGATATGGTTGAGTTCTGCAGGCGCATTGATCGC AACAGGGTTT MM007 (2^(nd) loop) (SEQ ID NO:103) CAAAAGCTGTGCCTGCAGAACACAACCTTACCCACTTTGTTTTTGCGTCG ACATCACGG and (SEQ ID NO:104) ACGCAAAAACAAAGTGGGTAAGGTTGTGTTCTGCAGGCACAGCTTTTGCA TGCACTATG MM009 (2^(nd) loop) (SEQ ID NO:105) CAAAAGCTGTGCCTGCCTGTTAACACCTACTCTTAACTGTTTTTGCGTCG ACATCACGG and (SEQ ID NO:106) ACGCAAAAACAGTTAAGAGTAGGTGTTAACAGGCAGGCACAGCTTTTGCA TGCACTATG MM010 (1^(st) loop) (SEQ ID NO:107) TCAATGCGCATGCGCTCTTCCAACTCATTCTAACTGTCGGTGTTCCGATA TGCGTCT and (SEQ ID NO:108) CGGAACACCGACAGTTAGAATGAGTTGGAAGAGCGCATGCGCATTGATCG CAACAGG MM010 (2^(nd) loop) (SEQ ID NO:109) CAAAAGCTGTGCCTGCGCGCTTCCTACACACTCTAACTGTTTTTGCGTCG ACATCACGG and (SEQ ID NO:110) ACGCAAAAACAGTTAGAGTGTGTAGGAAGCGCGCAGGCACAGCTTTTGCA TGCACTATG MM017 (2^(nd) loop) (SEQ ID NO:111) CAAAAGCTGTGCCTGCCCTTTAGGCCTTTGCCCACCTTGTTTTTGCGTCG ACATCACGG and (SEQ ID NO:112) ACGCAAAAACAAGGTGGGCAAAGGCCTAAAGGGCAGGCACAGCTTTTGCA TGCACTATG FGFps1 (2^(nd) loop) (SEQ ID NO:113) AAGCTGTATCTGCTGGAACATCGATTCTACACCTTGTTTTTGCGTCGACA TCACGG and (SEQ ID NO:114) ACGCAAAAACAAGGTGTAGAATCGATGTTCCAGCAGATACAGCTTTTGCA TGCACT FGFps2 (1^(st) loop) (SEQ ID NO:115) GCGATCAATGCATCTGTACTTGGATTGACAGTACTCCTTGTCGGTGTTCC GATATGCGTC and (SEQ ID NO:116) GGAACACCGACAAGGAGTACTGTCAATCCAAGTACAGATGCATTGATCGC AACAGGGTTT FGFps2 (2^(nd) loop) (SEQ ID NO:117) AAGCTGTATCTGCACATGGATCGATAGTACTCCTTGTTTTTGCGTCGACA TCACGG and (SEQ ID NO:118) ACGCAAAAACAAGGTGTAGAATCGATCCATGTGCAGATACAGCTTTTGCA TGCACT and FGFpsB (2^(nd) loop) (SEQ ID NO:119) AAGCTGTATCTGTACATGGATCGATTGGACACCTTGTTTTTGCGTCGACA TCACGG and (SEQ ID NO:120) ACGCAAAAACAAGGTGTCCAATCGATCCATGTACAGATACAGCTTTTGCA TGCACT 1A8 (2^(nd) loop) (SEQ ID NO:121) CAAAAGCTGCGCATGTGTTACTACAGATTGGATCGAATGTTTTTGCGTCG ACATCACGG and (SEQ ID NO:122) ACGCAAAAACATTCGATCCAATCTGTAGTAACACATGCGCAGCTTTTGCA TGCACTATG 1A12 (2^(nd) loop) (SEQ ID NO:123) CAAAAGCTGTGCCTGCCCAACACTTTGGACTCATATGTGTTTTTGCGTCG ACATCACGGAC and (SEQ ID NO:124) ACGCAAAAACACATATGAGTCCAAAGTGTTGGGCAGGCACAGCTTTTGCA TGCACTATGAC 1E11 (2^(nd) loop) (SEQ ID NO:125) CAAAAGCTGCGCATGTTACTACTCTCAATTCCACCAATGTTTTTGCGTCG ACATCACGG and (SEQ ID NO:126) ACGCAAAAACATTGGTGGAATTGAGAGTAGTAACATGCGCAGCTTTTGCA TGCACTATG TGFps1 (2^(nd) loop) (SEQ ID NO:127) CAAAAGCTGTCTTTGTCCGGAAAACGATAACGTTTCTCCTTGTAATTGCG TCGACATCACGGACTTCTG and (SEQ ID NO:128) TGTCGACGCAATTACAAGGAGAAACGTTATCGTTTTCCGGACAAAGACAG CTTTTGCATGCACTATGAC

The DNA sequences of the oligonucleotide pair used to make the cassette to introduce the MM021 peptide into the chymotrypsin reactive site loops of the p2JM103-Ink2-BBI expression vector are provided below. The cassette was ligated into the SphI and SalI restriction sites in the vector.

MM021 (2^(nd) loop) (SEQ ID NO:129) CAAAAGCTGTGCTTGTAAACACAACGTACGTCTTTTATGTTTTTGCG and (SEQ ID NO:130) TCGACGCAAAAACATAAAAGACGTACGTTGTGTTTACAAGCACAGCTTTT GCATG

Libraries made of cysteine constrained peptides are popular reagents (e.g. the commercially available PhD-C7C Phage Display Peptide Library Kit; NEB) for selecting peptides that bind to substrates of interest. BBI has two cysteine constrained reactive site loops that are structurally similar to the peptide loops displayed in various methods used to select peptide binders. So, once a cysteine constrained binding peptide has been selected, BBI is suitable for use as a scaffold to present the peptide in a binding reaction.

The VEGF binding peptide CK37281 (See e.g., co-pending U.S. Provisional Patent Application Ser. No. 60/520,403, filed Nov. 13, 2003, incorporated herein by reference) was grafted into BBI by replacing the trypsin, chymotrypsin, or both reactive site loops, with the CK37281 peptide sequence (ACYNLYGWTC) (SEQ ID NO:9) by using DNA oligonucleotide cassettes. To facilitate the construction, an EcoRI site was introduced in the coding region of the BBI gene (custom synthesized by Operon Technologies; See, Example 1) between the trypsin and chymotrypsin reactive site loops by QuikChange® site-directed mutagenesis, using methods described by the manufacturer (Stratagene) using the primers BowBeco-F and BowBeco-R, shown above (0.5 pmol of each primer was used in the QuikChange® reaction; after an initial denaturation step of 97° C. for 3 minutes, 18 PCR cycles of 68° C. for 12 minutes, 95° C. for 30 seconds and 55° C. for one minute, followed by a final extension reaction for 15 minutes at 68° C.).

To replace the trypsin inhibitory peptide loop, two DNA oligonucleotides (IBBCK81+ and IBBCk81−) were annealed and ligated into the SacI and EcoRI restriction sites. Likewise, to replace the chymotrypsin inhibitory peptide loop, EcoRI and SalI sites were used for insertion of a DNA cassette made by annealing the oligonucleotides (2BBck81+ and 2BBck81−). The CK37281 peptide was grafted into both loops by inserting the CK37281 peptide in the chymotrypsin loop (using the oligonucleotides (2BBck81+ and 2BBck81−) after the trypsin loop was first replaced by the CK37281 peptide. BBI with the CK37281 peptide in the trypsin loop (1BBIck81) was moved into the pJM103BBI expression vector as a SacI-SphI fragment. BBI with the CK37281 in the chymotrypsin loop (2BBIck81), or both loops (12BBIck81), was moved into pJM103BBI as SacI-SalI fragments. The correct sequences were verified by DNA sequencing (the sequence of 12BBIck81 gene is shown in FIG. 3). The resulting vectors, pJM103-1 BBIck81, pJM103-2BBIck81, or pJM103-12BBIck81, were used to transform B. subtilis BG3934comK, and the production of the BCE fusion proteins was determined as in Example 1 above.

The fusion protein running at ˜44 kDa was detected by SDS-PAGE to be the major protein present in the cell free broth. Although in some cases, there was significant degradation (up to 50%) of the BBI moiety (especially after >48 h of growth in MBD medium), as observed by the presence of a prominent protein band running at ˜34 kDa corresponding to the BCE103 catalytic core. In these cases, the titers of the BCE103 cellulase were similar to that measured with fusions to the wild-type BBI (Example 1), but the activity of the BBI (trypsin inhibition with 2BBIck81, or chymotrypsin inhibition with 1BBIck81) was generally about two fold less.

To reduce the proteolytic degradation of BBI variants during growth (i.e. decrease the amount of BCE103 cellulase core present on SDS-PAGE gels in comparison to the fusion protein), a Bacillus subtilis strain with nine protease genes deleted, BG6006 (degU^(Hy)32, oppA, ΔspoIIE3501, ΔaprE, ΔnprE, Δepr, ΔispA, Δbpr, Δvpr ΔwprA, Δmpr-ybjF, ΔnprB, amyE::xylRPxylAcomK-ermC), was used as an expression host, and the growth temperature (35° C.) and aeration (200 rpm) were reduced. With these changes, a major fusion protein band (˜44 kDa) was observed on SDS-PAGE gels with an insignificant band present at the molecular weight expected for the BCE catalytic core protein (˜34 kDa).

In addition to the CK37281 peptide, a number of other cysteine constrained peptides were produced when substituted into the trypsin and/or chymotrypsin reactive site loops of BBI fused to the C-terminus of the BCE103 cellulase. Specific examples included:

Peptides designed or selected as complement antagonists, compstatin introduced into the 1^(st) or 2^(nd) reactive site loops (See, Sahu et al., J. Immunol., 157: 884-891, [1996]), C2c (1^(st) loop), C3c (1^(st) loop), C4c (1St loop) and C5c (1^(st) loop); or peptides selected in a Factor B binding reaction 1B, 2B, 4A, 5A, 6-1A, 7A, 8B, 9A, 10B, 11-1A, 12B, 13A, and 15-1A (all in 2^(nd) loop);

Peptides designed to bind to the proteases Factor Xa or stratum corenum chymotrypsin, Xa1 (2^(nd) loop) or hSCC1 (1^(st) loop), respectively;

Peptides selected in FGF5 binding reactions 1A6 (1^(st) or 2nd loop), 1C2 (1^(st) or 2nd loop), 2E2 (1^(st) or 2nd loop), 2E5 (1^(st), 2^(nd) or both loops), FGFns (1^(st) or 2^(nd) loop), FGFkr (1^(st) or 2^(nd) loop), FGFhl (1^(st) or 2^(nd) loop), FGFgy (1^(st) or 2^(nd) loop), MM005 (1^(st) or 2^(nd) loop), MM007 (1^(st), 2^(nd) or both loops), MM009 (2^(nd) loop), MM010 (1^(st), 2^(nd) or both loops), MM017 (2^(nd) loop), FGFps1 (2^(nd) loop), FGFps2 (1^(st), 2^(nd) or both loops), and FGFpsB (2^(nd) loop); and

Peptides selected in TGFβ-L binding reactions 1A8 (2^(nd) loop), 1A12 (2^(nd) loop), 1E11 (2^(nd) loop), TGFps1 (2^(nd) loop), and MM021 (2^(nd) loop).

The oligonucleotides used to introduce these peptides into either the trypsin (1^(st) loop) or chymotrypsin (2^(nd) loop) reactive site loops, and methods used to graft these peptides into BBI, are provided above. In all cases, fusion proteins were produced as determined by SDS-PAGE gels. However, with some substituted peptides, the amount of intact fusion protein was increased by reducing the proteolytic degradation as described above for the CK37281 substituted peptide.

Example 3 Activation of BBI by Thiol Reducing/Oxidizing Agents

After growth, the activity of the BBI (by trypsin or chymotrypsin inhibition) is typically some 5-20 times lower than what would be expected from the activity of the BCE103 cellulase measured in the cell free supernatants (the two molecules should be present at a 1:1 molar ratio in the fusion protein). An increase in the activity of BBI (measured by either trypsin or chymotrypsin inhibition) in the BCE103-BBI fusion protein can be routinely obtained by adding □ME, typically concentrations of 1-4 mM added to the MBD growth medium about 14 h after inoculation. The trypsin or chymotrypsin inhibitory activity of BBI in the fusion protein is also lower than expected when binding peptides (e.g. VegF binding peptide CK37281) replace the chymotrypsin or trypsin reactive site loop, respectively. As with the wild-type BBI, the inhibitory activity can be increased by treatment with bME. Unexpectedly, other thiol reducing agents (e.g., cysteine, reduced glutathione, DL-dithiothreitol and Tris[2-carboxyethyl]phosphine) had small or negligible effects on the activation of BBI during growth in these experiments. Also, additions of antioxidants (e.g., ascorbic acid or DL-α-tocopherol acetate) or other adjuvants to the growth medium (e.g., isoleucine, soybean oil, Tween-80), or growth at 30° C. did not significantly improve the BCE103:BBI activity ratio.

Specifically, to determine the BBI activation during growth, cultures of B. subtilis BG6006 transformed with p2JM103-E3-2BBIck81 (See, Example 4, below) were grown in 40 ml MBD medium in 250 ml shake flasks at 37° C. for 13 h. Then, the thiol reducing agents indicated on the graph in FIG. 4 were added and cell supernatants harvested after 62 h of growth. The reagents 2-mercaptoethanol (BME), cysteine (Cys), reduced glutathione (Glut), and DL-dithiothreitol (DTT) were added to the growth medium to the final concentrations indicated on the graph provided in FIG. 4. Concentrations of 5 mM BME can result in better BCE103:BBI activity ratios but typically result in an overall decrease in both BCE103 and BBI titers (see FIG. 4), at least partially due to the reduction in bacterial growth caused by the added reagent. Titers of BCE103 and 2BBIck81 were determined using the assays described in Example 1.

BBI activation was also achieved after partial purification of the fusion proteins (e.g. BCE-Ink2-2BBIck81, see Example 4 below) by Q-Sepharose ion exchange chromatography.

The fusion protein was purified from cell free broth obtained from shake flasks or fermentor runs. The broth was filtered, diluted five to ten fold in water and the pH adjusted to pH 7.5-8.0. The diluted sample was loaded onto a column packed with Q-Sepharose resin (GE Healthcare). The column was washed with 50 mM Tris pH 7.5 and then washed again in the same buffer containing 300 mM NaCl. The fusion protein was eluted in the same buffer with 700 mM NaCl.

To activate the BBI, the pooled fusion protein fractions were diluted ten fold in Assay Buffer then treated with 2 mM BME and 0.2 mM oxidized glutathione (GSSG) with constant mixing on a stir plate or rocker platform for about 24 h at room temperature. The BBI could generally be activated to about 70-100% of the expected trypsin inhibitory activity based on the measured concentration of the BCE103 cellulase. Although the activation method outlined above generally yielded the best results, in some cases, in order to maximize the activation of a given sample, screens were performed in 96-well plates to determine the optimal conditions. Initially, the typical conditions screened were the dilution in Assay Buffer (e.g., a 2-50 fold dilution series), BME concentration (e.g., series between 0.5-5 mM) and oxidized glutathione concentration (e.g. 0 mM then a series of 1/20 to ½ the BME concentration).

The activation of the fusion protein BCE-Ink2-2BBIck81 is shown in FIG. 5. In this specific example, the fusion protein from a Q-Sepharose purification was diluted 1:10 in Dulbecco's PBS (Mediatech) with 0.005% TWEEN®-80. Beta-mercaptoethanol was added to a final concentration of 3 mM and incubated overnight at room temperature on a rocker. The sample was further incubated at room temperature for about 60 h with vigorous stirring on a magnetic stir plate. The titers of the BCE103 and 2BBIck81 (before and after βME treatment) were determined by cellulase assays and trypsin inhibitory assays, respectively.

In some embodiments, such as for activating BBI or it variants in cell free broth from large volume fermentations, it is desirable to reduce the dilution and βME concentration in the activation reaction. This can be accomplished by using higher concentrations of buffer (500 mM Tris pH 8.6), or changing to zwitterionic buffers such as CHES (also CAPS, Tricine, TAPS, and other suitable zwitterionic buffers). For example, cell free broth (or fusion protein fractions purified by ion exchange chromatography) was diluted 1:1 in 375 mM CHES pH 8.6 with 0.005% TWEEN®-80 then activated with 1 mM BME and 10 mM Na₂SO₃ and incubated with stirring at room temperature for about 24 h. BBI or its variants, as BCE103 cellulase fusion proteins, were routinely activated by this method to 70-100% of the expected value (based on BCE103 cellulase activities).

Example 4 Release of Free BBI/Variants by Cleavage of the BCE103-BBI Fusion Proteins

This Example describes experiments developed to release free BBI or its variants by cleavage of the BCE103-BBI fusion proteins.

The sequences of the DNA oligonucleotide pairs that were annealed and ligated into the BamHI and SacI sites of pJM103-BBI to generate potential cleavage sites during culture growth between the BCE103 catalytic domain and BBI are provided below.

BCEsubBBI (a subtilisin-type sensitive peptide sequence) (SEQ ID NO:131) GATCCAGGTGGAGCTGCTTTAGTTGACGATGAGAGCT and (SEQ ID NO:132) CTCATCGTCAACTAAAGCAGCTCCACCTG BCEcbdLBBI (a portion of the 1^(st) CBD) (SEQ ID NO:133) GATCCAGGTGAACCTGACCCAACTCCTCCATCTGATCCTGGAGAATACCC AGCTTGGGACGATGAGAGCT and (SEQ ID NO:134) CTCATCGTCCCAAGCTGGGTATTCTCCAGGATCAGATGGAGGAGTTGGGT CAGGTTCACCTG BCEproBBI (the entire pro peptide of BBI) (SEQ ID NO:135) GATCCGGCGAACCTGCGTCTGTCTAAGCTTGGCCTGCTTATGAAATCAGA CCATCAGCACAGCAATGACGATGAGAGCT and (SEQ ID NO:136) CTCATCGTCATTGCTGTGCTGATGGTCTGATTTCATAAGCAGGCCAAGCT TAGACAGACGCAGGTTCGCCG BCEshortproBBI (a C-terminal portion of the pro peptide of BBI) (SEQ ID NO:137) GATCCAAAATCAGACCATCAGCACAGCAATGACGATGAGAGCT and (SEQ ID NO:138) CTCATCGTCATTGCTGTGCTGATGGTCTGATTTTG

The sequences of the DNA oligonucleotide pair that was annealed and ligated into the BamHI and SacI sites of p2JM103-BBI to fuse BBI to the 2^(nd) CBD linker of BCE103 cellulase are provided below.

BCEcbdDBBI (SEQ ID NO:139) GATCCAGGAGAACCGGACCCAACGCCCCCAAGTGATCCAGGAGAGTATCC AGCATGGGATTCAAATCAAATTTACACAAATGAAATTGTGTATCATAACG GTCAGTTATGGCAAGCGAAATGGTGGACACAAAATCAAGAGCCAGGTGAC CCATACGGTCCGTGGGAACCACTCAAATCTGACCCAGATTCAGACGATGA GAGCT and (SEQ ID NO:140) CTCATCGTCTGAATCTGGGTCAGATTTGAGTGGTTCCCACGGACCGTATG GGTCACCTGGCTCTTGATTTTGTGTCCACCATTTCGCTTGCCATAACTGA CCGTTATGATACACAATTTCATTTGTGTAAATTTGATTTGAATCCCATGC TGGATACTCTCCTGGATCACTTGGGGGCGTTGGGTCCGGTTCTCCTG

The peptide sequences susceptible to acid cleavage between aspartic acid and proline residues are provided below.

Linker 1 - WGDPHY (SEQ ID NO:141) (Lidell et al., J. Biol. Chem. 278: 13944-51 [2003]) Linker 2 - DNNDPI (SEQ ID NO:142) (Segalas et al., FEBS Lett., 371: 171-175 [1995′9 ) Linker 3 - VVADPN (SEQ ID NO:143 (Kemperman et al., Appl. Env. Microbiol., 69: 1589-1597 [2003])

Oligonucleotide primers used to introduce a BssHII site into pJM103BBI by QuikChange® site-directed mutagenesis are provided below.

BCEbss-F (SEQ ID NO:144) 5′-TGGCGTTCAGCAACATGAGCGCGCAGGCTGATGATTA (SEQ ID NO:145) 5′-TAATCATCAGCCTGCGCGCTCATGTTGCTGAACGCCA

Sequences of the DNA oligonucleotides that were annealed as a cassette (SalI-HindIII) to introduce HindIII and XhoI sites after the stop codon of BBI, to introduce a PacI site after the LAT, and remove the original HindIII site are provided below.

BCEterm+ (SEQ ID NO:146) 5′-GACATCACGGACTTCTGCTATGAGCCATGTAAACCAAGCGAGGACGA TAAAGAGAACTAAAAGCTTAACTCGAGGTTAACAGAGGACGGATTTCCTG AAGGAAATCCGTTTTTTTATTTTTAATTAAG BCterm− (SEQ ID NO:147) 5′-AGCTCTTAATTAAAAATAAAAAAACGGATTTCCTTCAGGAAATCCGT CCTCTGTTAACCTCGAGTTAAGCTTTTAGTTCTCTTTATCGTCCTCGCTT GGTTTACATGGCTCATAGCAGAAGTCCGTGATG

PCR primers used to generate the acid labile linkers provided above (i.e., Linker 1, Linker 2, and Linker 3) inserted between the BCE103 catalytic domain and BBI are provided below.

BCE103coreBssHII_FW (SEQ ID NO:148) 5′-CAGCAACATGAGCGCGCAGGCTG linkerWGDPHY_RV (SEQ ID NO:149) 5′-ATCGTCTGGATCCGGATAGTGGGGGTCTCCCCAAGATGCTGATTCTC TTATTTTTTCC linkerDNNDPI_RV (SEQ ID NO:150) 5′-ATCGTCTGGATCCGGTATGGGATCATTGTTGTCAGATGCTGATTCTC TTATTTTTTCCC linkerVVADPN_RV (SEQ ID NO:151) 5′-ATCGTCTGGATCCGGGTTGGGATCTGCAACTACAGATGCTGATTCTC TTATTTTTTCCC

PCR primers used to generate the acid labile linkers provided above (i.e., Linker 1, Linker 2, and Linker 3) inserted into the 1^(st) CBD linker.

BCE103corePstI_FW (SEQ ID NO:152) GCATAAGGAT GAGTCATCTG CAGCG LplusWGDPHY_RV (SEQ ID NO:153) 5′-ATCGTCTGGATCCGGATAGTGGGGGTCTCCCCACGGTTCTCCTGGAT CAGATGGCGG LplusDNNDPI_RV (SEQ ID NO:154) 5′-ATCGTCTGGATCCGGTATGGGATCATTGTTGTCCGGTTCTCCTGGAT CAGATGGCGG LplusVVADPN_RV (SEQ ID NO:155) 5′-ATCGTCTGGATCCGGGTTGGGATCTGCAACTACCGGTTCTCCTGGAT CAGATGGCGG

Protein sequence of the acid labile linkers inserted between the BCE103 catalytic domain and BBI are provided below. The acid labile linkers are shown in bold type and the sequences from the first CBD domain are underlined.

Linker 1 BCE-WGDPHY- PDP-BBI (SEQ ID NO:156) Linker 2 BCE-DNNDPI- PDP-BBI (SEQ ID NO:157) Linker 3 BCE-VVADPN- PDP-BBI (SEQ ID NO:158) LinkerPlus 1 BCE-IPPSDPTPPSDPGEP WGDPHY- PDP-BBI (SEQ ID NO:159) LinkerPlus 2 BCE-IPPSDPTPPSDPGEP-DNNDPI- PDP-BBI (SEQ ID NO:160) LinkerPlus 3 BCE-IPPSDPTPPSDPGEP-VVADPN- PDP-BBI (SEQ ID NO:161)

The sequences of the DNA oligonucleotide pairs that were annealed and ligated into the BamHI and SacI sites of pJM103-BBI to generate potential cleavage sites between the BCE103 catalytic domain and BBI during the purification process are provided below.

BCEentBBI (Enteropeptidase cleavage site) (SEQ ID NO:162) GATCCAGGTGGAGACGACGATGACAAAGACGATGAGAGCT and (SEQ ID NO:163) CTCATCGTCTTTGTCATCGTCGTCTCCACCTG BCEgenen1BBI (Genenase I cleavage site) (SEQ ID NO:164) GATCCAGGTGCTGCTCATTACGACGATGAGAGCT and (SEQ ID NO:165) CTCATCGTCGTAATGAGCAGCACCTG

The sequences of the DNA oligonucleotide pairs that were annealed and ligated into the BamHI and SacI sites of pJM103-Ink2-1 BBIck81 to generate potential cleavage sites between the BCE103 catalytic domain and BBI during the purification process are provided below.

BCEfurinBBI (Furin/Blisterase cleavage site) (SEQ ID NO:166) GATCCACGTGCTAAAAGAGACGATGAGAGCT and (SEQ ID NO:167) CTCATCGTCTCTTTTAGCACGTG BCEgenen2BBI (Genenase I cleavage site) (SEQ ID NO:168) GATCCAGGCGCTGCACACTACAACGACGATGAGAGCT and (SEQ ID NO:169) CTCATCGTCGTTGTAGTGTGCAGCGCCTG BCEfleBBI (Mpr cleavage site) (SEQ ID NO:170) GATCCATTCCTTGAAGACGATGAGAGCT and (SEQ ID NO:171) CTCATCGTCTTCAAGGAATG

Sequences of the oligonucleotide primer pairs used to introduce the E and E3 linkers in Linker 2 by QuikChange site-directed mutagenensis (Stratagene) are provided below.

BCE-EInk-BBI (Mpr cleavage site) CCCATACCGGAGCCAGACGATGAGAGCTC (SEQ ID NO:172) and CATCGTCTGGCTCCGGTATGGGATCATTGTTG (SEQ ID NO:173)

The protein sequence of the E3 linker between the BCE103 catalytic domain and BBI was DNNDPIPEPDDESFNMPIPEP (SEQ ID NO:174). In this sequence, the E Linker is underlined and the sequence generated by faulty recombination in E. coli is shown in bold type. Cleavage by Mpr (or V8 protease) can occur after any of the three glutamic acids present in the E3 Linker. Thus, the structure was BCE-(SEQ ID NO:174)-BBI

The sequences of the DNA oligonucleotide pairs that were annealed and ligated into the BamHI and SacI sites of p2JM103-Ink2-2BBIck81 to generate potential Genenase I cleavage sites between the BCE103 catalytic domain and BBI are provided below.

BCEgenen3BBI (SEQ ID NO:175) GATCCAGGCGCTGCACACTACAAATCAGACCATCAGCACAGCAATGACGA TGAGAGCT and (SEQ ID NO:176) CTCATCGTCATTGCTGTGCTGATGGTCTGATTTGTAGTGTGCAGCGCCTG BCEgenen4BBI (SEQ ID NO:177) GATCCAGGCGCTGCACACTACGTAGAATTTCAAGACGATGAGAGCT and (SEQ ID NO:178) CTCATCGTCTTGAAATTCTACGTAGTGTGCAGCGCCTG

The protein sequence of a Genenase I sensitive cleavage site (also acid and Mpr sensitive) inserted between the BCE103 catalytic domain and BBI was DNNDPIPDPGAAHYVEFQ (SEQ ID NO:179). The Genenase I site (Gen4 Linker) is in bold type (cleavage occurs between the tyrosine and valine) (NEB) and Linker 2 is underlined. Cleavage by Mpr can also occur after the glutamic acid that follows the valine in the Gen4 linker. The sequence used herein was BCE-SEQ ID NO:179)-BBI

Cleavage sites in the BCE103-Ink2-2BBIck81 fusion protein are indicated below. The C-terminal seven amino acids of the BCE103 catalytic domain (underlined), linker 2 sequence (bold type), and 2BBIck81 sequences are shown. The acid/heat labile Asp-Pro bonds are indicated with solid headed arrows and the Mpr sensitive bonds after glutamic acids are indicated with line headed arrows.

In order to isolate free BBI or its variants, the BBI moiety needs to be cleaved from the BCE103-BBI fusion protein. In some embodiments, this is accomplished during growth, by proteases intrinsically produced by B. subtilis. In some alternative embodiments, this cleavage occurs after growth, during the purification process (e.g. by acid/heat or proteolytic cleavage). Linkers potentially susceptible to cleavage during growth were designed (See, above, sub, cbdL, pro, shortpro, and cbdD) and cloned into the pJM103BBI or p2JM103BBI expression vectors as BamHI-SacI cassettes. The production of fusion protein versus BCE103 catalytic domain was analyzed on SDS-PAGE gels as described in Example 1.

Little cleavage of the fusion protein was observed for all these linkers except with the pro linker, which was nearly completely cleaved so that very little intact fusion protein was observed on gels, although there was a large band corresponding to the BCE103 catalytic core. Unfortunately, this cleavage during growth resulted in negligible BBI activity measured in cell free supernatants and no BBI band could be identified on SDS-PAGE gels. Although it is not intended that the present invention be limited to any particular mechanism or theory, it is possible that the BBI is particularly sensitive to proteolytic degradation in its inactive form. Thus, cleavage during the purification process after activation is generally preferred.

In some embodiments, the bonds between aspartic acid and proline residues are cleaved by heat treatment at acidic pH as known in the art (See e.g., Landon, Meth. Enzymol., 47:145-149 [1977]). The 1st CBD linker in the BCE103 cellulase has three Asp-Pro dipeptide sequences (See, FIG. 1) with the potential to be cleaved by acid/heat treatment. However, cleavage by acid/heat treatment at these sites was found to be inefficient. Protein sequences that are especially labile to acid/heat have been described in the literature, three of such sequences are WGDPHY (SEQ ID NO:141), DNNDPI (SEQ ID NO:142), and VVADPN (SEQ ID NO:143) (i.e., Linkers 1, 2 and 3).

Before these acid labile linkers were introduced into the BCE103-BBI expression vector, pJM103BBI, a BssHII site was introduced by QuikChange® XL (Stratagene) mutagenesis (using the manufacturer's methods; and described in Example 2 above, except 8 minute extension and 1 minute denaturation steps were used) in the aprE signal sequence coding region using the oligonucleotide primers BCEbss-F and BCEbss-R (provided above). Then, HindIII and XhoI sites were inserted in front of the LAT terminator (after the BBI stop codon) and a PacI site was added after the terminator (the original HindIII site after the LAT terminator was removed) by inserting an oligonucleotide cassette (BCEterm+ and BCEterm−; provided above) into the SalI and the original HindIII sites. This new vector was called “p2JM103BBI.”

The acid labile linker fragments were generated by PCR, using forward primer BCE103coreBssHII_FW with each of the reverse primers, linker WGDPHY_R, linker DNNDPI_RV, or linker VVADPN_RV (the sequences of which are all provided above) and p2JM103BBI as the template (see Example 1 for the PCR protocol). The PCR fragments of 970 bp were digested with BamHI and PstI, the 154 bp fragments encoding the acid linker fragments were isolated from an agarose gel after electrophoresis, and ligated into the p2JM103 vector digested with BamHI and PstI that had also been purified from a gel. The linker sequences in the final expression vectors, p2JM103Ink1-BBI, p2JM103Ink2-BBI and p2JM103Ink3-BBI, were verified by DNA sequencing.

Competent B. subtilis strain BG3934comK or BG6006 were transformed with the plasmids, colonies selected on 5 μg/ml chloramphenicol LA plates and amplified to 25 μg/ml chloramphenicol as described in Example 1.

Similarly, the acid labile linkers were inserted into the first CBD linker. Specifically, PCR fragments were generated using the forward primer BCE103corePstI_FW with the reverse primers LplusWGDPHY_RV, LplusDNNDPI_RV, or LplusVVADPN_RV (See above, for the sequences) with p2JM103BBI as a template. The PCR fragments of about 150 bp were digested with BamHI and PstI, purified and ligated to the p2JM103BBI vector digested with BamHI and PstI. The correct sequences were verified by DNA sequencing and the plasmids p2JM103pIInk1-BBI, p2JM103pIInk2-BBI and p2JM103pIInk3-BBI were used to transform B. subtilis strains as described above.

After growth in MBD medium, the fusion proteins were purified by ion exchange chromatography essentially as described above (See, Example 2). The fusion protein was cleaved by treatment at 55° C. for 16 h in 10% formic acid. The BCE103 catalytic domain precipitated during the acid treatment and was removed by centrifugation. The free BBI in the supernatant was dried overnight on a SpeedVac. The sample was suspended in 50 mM Tris pH 8 before loading on the SDS-PAGE gel. By analysis of the protein stained SDS-PAGE gels, it was observed that acid cleavage was much more efficient in the fusion proteins where Linker 2 was inserted between the BCE103 catalytic domain and BBI (BCE-DNNDPI-PDP-BBI). This linker was found to be cleaved in a couple of hours at 75° C. in 20 mM glycine pH 2.

In alternative embodiments, the fusion protein was cleaved by treatment with a protease during the purification process. Linkers were designed with cleavage sites for glutamic acid specific proteases (e.g., Mpr or V8 protease), Furin/blisterase, Genenase I, and Enteropeptidase (Enterokinase). These linkers were introduced as oligonucleotide cassettes (See above, for the sequences) between the BCE103 catalytic core and BBI in the expression vector using the BamHI and SacI sites (See, FIG. 1). In the coding region of the original expression vector (pJM103BBI), there is a glutamic acid residue in the 1^(st) CBD domain and at the third residue in BBI (See, FIG. 1), which is contemplated to be susceptible to cleavage by glutamic acid specific proteases such as B. subtilis Mpr (BsMpr) or V8 protease. However, neither BsMpr nor V8 protease were found to cleave the BCE-BBI fusion protein very efficiently at these sites. Thus, it was necessary to design other linkers that were susceptible to cleavage by these proteases.

The six acid labile linkers described above were tested for cleavage by BsMpr. These fusion proteins were cleaved by treatment for 16 h with 16 μg of BsMpr at room temperature. After cleavage, the BCE103 catalytic domain was precipitated by the addition of 10% formic acid and removed by centrifugation. The free BBI in the supernatant was dried overnight on a SpeedVac. The sample was suspended in 50 mM Tris pH 8, before loading on the SDS-PAGE. Similar to the acid cleavage, the BCE-DNNDPI-PDP-BBI (Linker 2) fusion protein was much more efficiently cleaved by BsMpr than any of the other linkers. Therefore, BBI and its variants were found to be effectively released from the BCE-DNNDPI-PDP-BBI fusion protein either by acid/heat treatment or proteolytic digestion with a glutamic acid specific protease such as BsMpr. Several other linkers designed for cleaved by Mpr (e.g., E, E3 linker, and fle, provided above) were tested but none of them had any advantages over Linker 2 (the E3 linker was generated by faulty recombination in E. coli after transformation with the QuikChange® site-directed mutagenesis reaction designed to construct the E linker). As shown above, there are two acid/heat labile cleavage sites in Linker 2 and three sites sensitive to cleavage by Mpr.

Linkers designed for cleavage by Furin or Blisterase (NEB) (BCEfurinBBI), or Enteropeptidase (Enterokinase, NEB) (BCEentBBI) were tested, but none of these sequences were cleaved efficiently by the appropriate protease. Four linkers were also designed (BCEgenen1BBI, BCEgenen2BBI, BCEgenen3BBI, and BCEgenen4BBI) and tested for cleavage by Genenase I (NEB). Efficient cleavage of the fusion protein was observed only with the Gen4 Linker (BCEgenen4BBI). BsMpr was also found to efficiently cleave the Gen4 linker.

After activation of the purified BCE-Ink2-2BBIck81 fusion protein, cleavage by BsMpr does not go to completion as judged by SDS-PAGE gels. However, it was discovered that complete cleavage after activation of BCE-BBI fusion proteins with Linker 2 (or the Gen4 linker) can be accomplished by using the Mpr protease isolated from Bacillus licheniformis (BIMpr). While it is not intended that the present invention be limited to any particular mechanism, cleavage after the third amino acid in mature BBI appeared to be more sensitive to BIMpr while cleavage after the sixth amino acid from the C-terminus of BBI is more sensitive to BsMpr cleavage.

In some embodiments, after cleavage, the BBI is purified away from the BCE103 catalytic domain by selective acid precipitation (pH 3 or lower) of the BCE103 catalytic domain as described above, ion exchange chromatography (See, Example 5), or by selective binding of BBI on an anhydrotrypsin-agarose (Sigma) column loaded in 50 mM Tris pH 8.0, washed with 50 mM Tris pH 8.0 with 150 mM NaCl, then eluting bound BBI with 50 mM glycine pH 2.2 with 300 mM NaCl).

Example 5 Binding of BBIck81 to VegF

In this Example, experiments conducted to assess the binding of BBIck81 to VegF are described. The BCE103-Ink2-2BBIck81 fusion protein was produced in B. subtilis as described in Example 2. The fusion protein was purified, and the BBI trypsin inhibitory activity was increased by treatment with βME and oxidized glutathione as described in Example 3. The fusion protein was cleaved by BsMpr protease (See, Example 4) and the free 2BBIck81 was purified from the BCE103 catalytic domain by ion exchange chromatography using a Q-Sepharose column.

Briefly, after cleavage, the pH of the cleaved sample was adjusted to 5.5, the sample was then loaded onto the column (equilibrated with 25 mM MES pH 5.5). The free 2BBIck81 was washed through the column using 25 mM sodium acetate pH 5.0 while the BCE103 catalytic core remained bound to the resin. The 2BBIck81 fraction was concentrated by ultrafiltration and analyzed using an electrochemiluminescence (ECL) based binding assay (BioVeris). The Anti-VegF antibody (Santa Cruz) and VegF (PeproTech) were labeled with the electrochemiluminescent dye and biotin, respectively, as described by the manufacturer (BioVeris). All materials were in Dulbecco's PBS (Mediatech) supplemented with 0.1% TWEEN®-80. An initial dilution series of Anti-VegF antibody (125, 250 and 500 ng/ml) and VegF (100, 150, 200 and 250 ng/ml) were tested in the binding assay to determine the concentrations of each that would give a robust ECL signal.

For testing 2BBIck81 binding, 50 μL of 500 ng/ml ECL labeled Anti-VegF antibody, 50 μL of 250 ng/ml biotinylated VegF and 100 μL 2BBIck81 (series of 12.5, 15, 31.25, 62.5, 125, 250 or 500 ng/ml) were incubated at room temperature for 2 h with shaking. Then, 50 μL of 0.2 mg/ml streptavidin coated beads were added and the reaction was incubated at room temperature for 30 minutes. The ECL signal was measured using a BioVeris M8/384 Analyzer as described by the manufacturer (BioVeris). As shown in FIG. 6, the ECL signal decreased as increasing concentrations of 2BBIck81 displaced more of the labeled Anti-VegF antibody bound to VegF attached to the magnetic beads.

Thus, the CK37281 peptide when grafted onto the chymotrypsin inhibitory loop of BBI (2BBIck81) competed with the Anti-VegF antibody for binding to VegF at micromolar concentrations. In fact, 2BBIck81 competed for VegF binding better than the synthesized CK37281 peptide itself (See, FIG. 6). The CK37281 peptide inserted into the trypsin inhibitory loop, 1BBIck81, also competed with the Anti-VegF antibody in the BioVeris assay. Thus, BBI was found to be useful as a scaffold to present active binding peptides selected by various screening methods.

Example 6 Use of Alternative Fusion Partners for the Production of 2BBIck81

In this Example, experiments conducted to evaluate alternative fusion partners are described. The DNA sequence of the oligonucleotide primers used to amplify the dsbC gene (E. coli) from pET-40b(+) are provided below. These primers generate a BssHII site at the 5′ end and a BamHI at the 3′ end for cloning into p2JM103-Gen4-2BBIck81.

DsbCBBI-F (SEQ ID NO:181) AACATGAGCGCGCAGGCTGATGACGCGGCAATTCAACAAACGTTAG DsbCBBI-R (SEQ ID NO:182) TCGTCTGGATCCGGTATGGGATCATTGTTGTCACCAGAACCACTAGTTGA TCCTTTACCGCTGGTCATTTTTTGGTG

The DNA sequences of the oligonucleotides that were annealed together to make a cassette (Alw44I-BamHI) for fusing the P. mendocina cutinase gene to BBI with Linker 2, are provided below.

CutinaseBBI+ (SEQ ID NO:183) TGCACTTCTCTGCTTTGGTCTGTTGAACGCAGAGGTCTTGACAACAATGA TCCTATTCCG CutinaseBBI− (SEQ ID NO:184) GATCCGGAATAGGATCATTGTTGTCAAGACCTCTGCGTTCAACAGACCAA AGCAGAGAAG

Because the BBI moiety has seven disulfide bonds, it is contemplated that higher titers of active BBI will be obtained using fusion proteins other than the BCE103 cellulase catalytic domain. For example, in some embodiments, compositions such as thiol-disulfide oxidoreductases and/or protein disulfide isomerases find use as fusion proteins to help produce correctly folded BBI moieties. In this embodiment, no additional activation step is needed under most circumstances. In additional embodiments, other proteins produced at high titers in B. subtilis also find use as fusion partners. For example, the thermostable protein disulfide isomerase from the fungus Humicola insolens (hiPDI) has been used as a fusion partner to produce the light chain of immunoglobulin G (2 disulfides) in Bacillus brevis (See, Kajino et al., Appl. Env. Microbiol., 66:638-642 [2000]).

To determine whether hiPDI could be a better fusion partner than BCE103 for the production of BBI, this hiPDI gene was synthesized (DNA2.0) and cloned into the expression vector, p2JM103-Ink2-2BBIck81 (See, Example 4) as a BssHII-SacI fragment. In designing the synthetic gene, codons occurring with high frequency in highly expressed B. subtilis genes were selected except in cases where restriction sites were introduced or deleted. In the final construction, the N-terminus of the mature hiPDI gene was fused to the AprE signal sequence and the C-terminus was fused to a linker with an Enteropeptidase cleavage site (Kajino et al., Appl. Env. Microbiol., 66:638-642 [2000]), which in turn was fused to 2BBIck81 (See, FIG. 7). This expression vector, p2JM-PDI-EK-2BBIck81, was used to transform B. subtilis BG6006 and the production of the fusion protein was determined in MBD medium (as described in Example 1) with or without 2 mM BME added 14 h after inoculation.

As determined by SDS-PAGE gels, the production of the PDI-2BBIck81 fusion protein was typically somewhat less than the BCE-2BBck81 grown under identical conditions. The BBI titers (trypsin inhibition) measured from the PDI-2BBIck81 cell free supernatants were also typically less than the BCE-2BBIck81 fusion. As with fusions to BCE103, the measured activities of BBI when fused to PDI were higher when grown in 2 mM BME and the BBI activity was increased by the addition of BME to the cell free supernatants after growth when grown in BME free medium (as described in Example 3). Thus, the thiol-disulfide oxidoreductase activity of PDI does not seem to significantly improve the titers of active 2BBIck81 in the fusion protein or obviate the need for activation of the BBI molecule.

In order to increase the reduction potential of the fusion protein, which was contemplated to improve the BBI titers during growth, DsbC from Escherichia coliwas used as a fusion partner for 2BBIck81. The dsbC gene was amplified by PCR using Herculase Enhanced DNA polymerase as described by the manufacturer (Stratagene) using DsbCBBI-F and DsbCBBI-R as primers (sequences shown above) and pET-40b(+) (Novagen) as a template. The isolated PCR fragment was cloned into the vector p2JM103-Gen4-2BBIck81 (See, Example 4) as a BssHII-BamHI fragment. The correct sequence of the fusion gene was verified by DNA sequencing. In this case, the titers of the DsbC-2BBIck81 fusion protein were significantly lower than the BCE-2BBIck81 fusion protein as judged on SDS-PAGE gels and the titers of the active 2BBIck81 measured by trypsin inhibition were much lower as well.

Other proteins that are produced at high titers in B. subtilis find use as fusion partners for the production of BBI. One such protein is the cutinase from Pseudomonas mendocina, which has been expressed at high titers utilizing the aprE promoter from B. subtilis (See e.g., U.S. Pat. No. 5,429,950, herein incorporated by reference). The aprE-cutinase gene fusion as an EcoRI-Alw44I fragment (from pAK-15) was ligated with an Alw44I-BamHI linker oligonucleotide cassette (See, sequence above) into the p2JM103-Ink2-2BBIck81 (See, Example 4) that had been cut with EcoRI and BamHI. This cutinase-linker2-2BBIck81 expression vector (See, FIG. 8 for the EcoRI-BamHI aprE-cutinase-linker2 sequence) was used to transform B. subtilis BG6006 cells and the fusion protein was produced in MBD medium as described previously for the other fusion proteins (See, Example 1). In this case, the cutinase-linker2-2BBIck81 fusion protein was not the major band observed on SDS-PAGE gels and the measured lipase titers (as measured using the methods provided in U.S. Pat. No. 5,429,950) and BBI titers were much less (ca. 20 fold) than found with the BCE-2BBIck81 fusion protein. Also, the BBI titers in the cutinase fusion protein were not improved significantly when 3 mM □ME was added to the growth medium. Thus, the highest titers of active 2BBIck81 was consistently obtained by activation of the BCE-2BBIck81 fusion protein. Nonetheless, it is contemplated that various fusion partners will find use in the present invention.

Example 7 Expression of Truncated BBI-AV BBIt-AV; SEQ ID NO:187

This Example describes experiments that were performed to improve the production of the 2BBIck81 BBI protein (BBI-AV; SEQ ID NO:186) DDESSKPCCDQCACTKSNPPQCRCSDMRLNSCHSACKSCACYNLYGWTCFCVDITDFCYEPCKPSEDDKEN (SEQ ID NO:18).

The Examples above and the journal article of Vogtentanz et al., 2007 (Protein Expr Purif 55:40-52 [2007]) describe a method for producing a fusion protein that comprises the soybean Bowman-Birk protease inhibitor (BBI) fused to the C-terminus of the BCE103 cellulase catalytic domain (BCE). As described above, this system has been used to produce BBI molecules with various variant peptides replacing the trypsin and/or chymotrypsin inhibitory loops. For example, a BBI molecule, BBI-AV (SEQ ID NO:186) containing the VEGF binding peptide, CK37281 (ACYNLYGWTC; SEQ ID NO:9), that replaces the native chymotrypsin inhibitory loop (ICALSYPAQC; SEQ ID NO:388) was produced, purified and shown to compete in an electrochemiluminescent (ECL) based binding assay (BioVeris) with a monoclonal antibody for binding to VEGF. However, the recovered yield of the variant BBI-AV was less than that of the wild-type BBI most likely due to an initial higher percentage of molecules with incorrectly formed disulfide bonds.

A truncated form of the BBI-AV of SEQ ID NO:186 (i.e. BBIt-AV of SEQ ID NO:187) DDESSKPCCDQCACTKSNPPQCRCSDMRLNSCHSACKSCACYNLYGWTCFCVDITDFCYEPCKPSE (SEQ ID NO:187), which lacks the 5 C-terminal amino acids of SEQ ID NO:186 was shown to bind better than the full-length molecule (i.e. untruncated molecule) to VEGF in an ECL (BioVeris) assay using a labeled monoclonal antibody against VEGF as a competitor. The C-truncated version of BBI-AV, called BBIt-AV (SEQ ID NO:187), can be produced during the purification process by trimming the C-terminus with GluBL protease (glutamyl endopeptidase I from Bacillus licheniformis). Alternatively, the BBIt-AV can be obtained by introducing a stop codon using an engineered oligonucleotide cassette as follows.

The following BBI-trunc+ and BBI-trunc− oligonucleotides were annealed to generate an oligonucleotide cassette

BBI-trunc+ (SEQ ID NO:188) 5′-TCGACATCACGGACTTCTGCTATGAACCATGTAAACCAAGCGAGTAA A and (SEQ ID NO:189) 5′-AGCTTTTACTCGCTTGGTTTACATGGTTCATAGCAGAAGTCCGTGAT G

The oligonucleotide cassette was then ligated into the SalI and HindIII sites of the expression vector p2JM103-Ink2-2BBIck81, and the correct sequence was verified and the resulting expression vector was called p2JM103-Ink2-BBIt-AV.

To improve the expression, a polynucleotide sequence (SEQ ID NO:190 gctggtaaa) encoding the first three amino acids of the AprE pro-peptide, AGK (SEQ ID NO:191) (U.S. Pat. No. 5,429,950), was inserted between the end of the AprE signal sequence and the start of the mature BCE103 cellulase using a QuikChange® site-directed mutagenesis kit (Stratagene). The site directed mutagenesis was performed essentially as described by the manufacturer using p2JM103-Ink2-BBIt-AV as a template with the oligonucleotide primers:

BCE-AGK-F: (SEQ ID NO:192) 5′-GCGCGCAGGCAGCTGGTAAAGATGATTATTCAGTTGTAGA and (SEQ ID NO:193) 5′-AATAATCATCTTTACCAGCTGCCTGCGCGCTCATGTTGCT

The correct insertion was verified by DNA sequencing and the resulting expression vector was called p2JMagk103-Ink2-BBIt-AV (SEQ ID NO:194).

(SEQ ID NO:194) GAATTCTCCATTTTCTTCTGCTATCAAAATAACAGACTCGTGATTTTCCA AACGAGCTTTCAAAAAAGCCTCTGCCCCTTGCAAATCGGATGCCTGTCTA TAAAATTCCCGATATTGGTTAAACAGCGGCGCAATGGCGGCCGCATCTGA TGTCTTTGCTTGGCGAATGTTCATCTTATTTCTTCCTCCCTCTCAATAAT TTTTTCATTCTATCCCTTTTCTGTAAAGTTTATTTTTCAGAATACTTTTA TCATCATGCTTTGAAAAAATATCACGATAATATCCATTGTTCTCACGGAA GCACACGCAGGTCATTTGAACGAATTTTTTCGACAGGAATTTGCCGGGAC TCAGGAGCATTTAACCTAAAAAAGCATGACATTTCAGCATAATGAACATT TACTCATGTCTATTTTCGTTCTTTTCTGTATGAAAATAGTTATTTCGAGT CTCTACGGAAATAGCGAGAGATGATATACCTAAATAGAGATAAAATCATC TCAAAAAAATGGGTCTACTAAAATATTATTCCATCTATTACAATAAATTC ACAGAATAGTCTTTTAAGTAAGTCTACTCTGATTTTTTTAAAAGGAGAGG GTAAAGAGTGAGAAGCAAAAAATTGTGGATCAGCTTGTTGTTTGCGTTAA CGTTAATCTTTACGATGGCGTTCAGCAACATGAGCGCGCAGGCAGCTGGT AAAGATGATTATTCAGTTGTAGAGGAACATGGGCAACTAAGTATTAGTAA CGGTGAATTAGTCAATGAACGAGGCGAACAAGTTCAGTTAAAAGGGATGA GTTCCCATGGTTTGCAATGGTACGGTCAATTTGTAAACTATGAAAGCATG AAATGGCTAAGAGATGATTGGGGAATAACTGTATTCCGAGCAGCAATGTA TACCTCTTCAGGAGGATATATTGACGATCCATCAGTAAAGGAAAAAGTAA AAGAGACTGTTGAGGCTGCGATAGACCTTGGCATATATGTGATCATTGAT TGGCATATCCTTTCAGACAATGACCCGAATATATATAAAGAAGAAGCGAA GGATTTCTTTGATGAAATGTCAGAGTTGTATGGAGACTATCCGAATGTGA TATACGAAATTGCAAATGAACCGAATGGTAGTGATGTTACGTGGGACAAT CAAATAAAACCGTATGCAGAAGAAGTGATTCCGGTTATTCGTGACAATGA CCCTAATAACATTGTTATTGTAGGTACAGGTACATGGAGTCAGGATGTCC ATCATGCAGCCGATAATCAGCTTGCAGATCCTAACGTCATGTATGCATTT CATTTTTATGCAGGAACACATGGACAAAATTTACGAGACCAAGTAGATTA TGCATTAGATCAAGGAGCAGCGATATTTGTTAGTGAATGGGGGACAAGTG CAGCTACAGGTGATGGTGGTGTGTTTTTAGATGAAGCACAAGTGTGGATT GACTTTATGGATGAAAGAAATTTAAGCTGGGCCAACTGGTCTCTAACGCA TAAGGATGAGTCATCTGCAGCGTTAATGCCAGGTGCAAATCCAACTGGTG GTTGGACAGAGGCTGAACTATCTCCATCTGGTACATTTGTGAGGGAAAAA ATAAGAGAATCAGCATCTGACAACAATGATCCCATACCGGATCCAGACGA TGAGAGCTCTAAACCCTGTTGCGATCAATGCGCATGTACGAAATCAAATC CTCCACAGTGTCGGTGTTCCGATATGCGTCTGAATTCCTGTCATAGTGCC TGCAAAAGCTGCGCATGTTATAACCTGTACGGGTGGACCTGTTTTTGCGT CGACATCACGGACTTCTGCTATGAACCATGTAAACCAAGCGAGTAAAAGC TTAACTCGAGGTTAACAGAGGACGGATTTCCTGAAGGAAATCCGTTTTTT TATTTTTAATTAAGAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTG TGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAATAA AGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCG TTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCA TTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCT CTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGG CGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATC AGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCA GGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCC CCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCC GACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGC GCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTC CCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAG TTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCG TTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAAC CCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGAT TAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGC CTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTG AAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACA AACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGC GCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCT GACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATT ATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTA AATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGC TTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCAT AGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTAC CATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCT CCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAG TGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGG AAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCC ATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATT CAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGT GCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAG TTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCT TACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAA CCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCG GCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCT CATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGC TGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCA GCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCA AAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCA TACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTC ATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGT TCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTA TTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGT CTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCC GGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCC GTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTAT GCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATAGATCTGGAGCTGT AATATAAAAACCTTCTTCAACTAACGGGGCAGGTTAGTGACATTAGAAAA CCGACTGTAAAAAGTACAGTCGGCATTATCTCATATTATAAAAGCCAGTC ATTAGGCCTATCTGACAATTCCTGAATAGAGTTCATAAACAATCCTGCAT GATAACCATCACAAACAGAATGATGTACCTGTAAAGATAGCGGTAAATAT ATTGAATTACCTTTATTAATGAATTTTCCTGCTGTAATAATGGGTAGAAG GTAATTACTATTATTATTGATATTTAAGTTAAACCCAGTAAATGAAGTCC ATGGAATAATAGAAAGAGAAAAAGCATTTTCAGGTATAGGTGTTTTGGGA AACAATTTCCCCGAACCATTATATTTCTCTACATCAGAAAGGTATAAATC ATAAAACTCTTTGAAGTCATTCTTTACAGGAGTCCAAATACCAGAGAATG TTTTAGATACACCATCAAAAATTGTATAAAGTGGCTCTAACTTATCCCAA TAACCTAACTCTCCGTCGCTATTGTAACCAGTTCTAAAAGCTGTATTTGA GTTTATCACCCTTGTCACTAAGAAAATAAATGCAGGGTAAAATTTATATC CTTCTTGTTTTATGTTTCGGTATAAAACACTAATATCAATTTCTGTGGTT ATACTAAAAGTCGTTTGTTGGTTCAAATAATGATTAAATATCTCTTTTCT CTTCCAATTGTCTAAATCAATTTTATTAAAGTTCATTTGATATGCCTCCT AAATTTTTATCTAAAGTGAATTTAGGAGGCTTACTTGTCTGCTTTCTTCA TTAGAATCAATCCTTTTTTAAAAGTCAATATTACTGTAACATAAATATAT ATTTTAAAAATATCCCACTTTATCCAATTTTCGTTTGTTGAACTAATGGG TGCTTTAGTTGAAGAATAAAAGACCACATTAAAAAATGTGGTCTTTTGTG TTTTTTTAAAGGATTTGAGCGTAGCGAAAAATCCTTTTCTTTCTTATCTT GATAATAAGGGTAACTATTGCCGGATCGTCCTCAGGAGTAGGCGACATCG CTAAATAATGATCTATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGA AAATACCGCATCAGGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGA AGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGG GATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCA CGACGTTGTAAAACGACGGCCAGT

The p2JMagk103-Ink2-BBIt-AV expression vector was used to transform bacterial cells to express BBIt-AV fused to the BCE protein (SEQ ID NO:195)

(SEQ ID NO:195) AGKDDYSVVEEHGQLSISNGELVNERGEQVQLKGMSSHGLQWYGQFVNYE SMKWLRDDWGITVFRAAMYTSSGGYIDDPSVKEKVKETVEAAIDLGIYVI IDWHILSDNDPNIYKEEAKDFFDEMSILYGDYPNVIYEIANEPNGSDVTW DNQIKPYAEEVIPVIRDNDPNNIVIVGTGTWSQDVHHAADNQLADPNVMY AFHFYAGTHGQNLRDGVDYALDQGAAIFVSEWGTSAATGDGGVFLDEAQV WIDFMDERNLSWANWSLTHKDESSAALMPGANPTGGWTEAELSPSGTFVR EKIRESASDNNDPIPDPDDESSKPCCDQCACTKSNPPQCRCSDMRLNSCH SACKSCACYNLGWTCFCVDITDFCYEPCKPSE

Example 8 BBIt-AV Site Saturation Libraries

This example describes experiments that were performed to generate modified variant BBPIs derived from the unmodified BBIt-AV by constructing site saturation libraries comprising substitutions at each of the 39 amino acid positions of the BBIt-AV molecule (SEQ ID NO:187) shown in FIG. 9.

BBIt-AV site-saturation libraries were created essentially as described by Amin et al. (Biotechniques 35: 1134-1140, [2003]) using a modified version of the QuikChange multi site-directed mutagenesis (QCMS) kit from Stratagene (La Jolla, Calif.) as follows. Overlapping forward and reverse primers with the NNS codon in the middle and 17-20 flanking bases were designed for each chosen site, and the sequences for each forward (F) and reverse (R) primer at each of the 66 amino acid positions in the BBI-AV molecule are shown in Table 1. Each mutagenesis reaction contained 50-100 ng template plasmid (p2JMagk103-Ink2-BBIt-AV; SEQ ID NO:194), 0.5 μl forward primer (25 μM), 0.5 μl reverse primer (25 μM), 1 μl dNTP's (QCMS kit), 2.5 μl 10×QCMS reaction buffer, 18.5 μl deionized water, and 1 μl of enzyme blend (QCMS kit), for a total volume of 25 μl. For BBI-AV libraries at residues 10, 11, 13 and 25, only 1 μL of the forward primer (25 □M) was used without the reverse primer. For libraries at residues 6, 37, 38, 50 and 53, the mutagenesis reaction was carried out with 0.5 μl of the forward mutagenic primer in combination with another primer (5′-CTATGCGGCATCAGAGCAGATTGTAC; SEQ ID NO:328 complementary to a sequence in the vector. For the mutagenesis reaction, the thermocycler program used was 1 cycle at 95° for 2 min., followed by 29 cycles of 95° C. for 30 sec, 55° C. for 1 min., and 68° C. for 10 minutes (MJ Research thermocycler). The template DNA was digested by the addition of 0.5-1 μl DpnI (from the QCMS kit) and incubation at 37° C. for 1-4 hours, followed by another addition of 0.5-1 μl DpnI and another incubation at 37° C. for 1-4 hours. The template DNA was obtained from E. coli dam⁺ strains, such as TOP10 (Invitrogen, Carlsbad, Calif., USA), to ensure that it would be susceptible to Dpn I digestion.

For efficient transformation of B. subtilis, the DNA in each mutagenesis reaction was amplified by rolling circle amplification (RCA) using the Templiphi kit from Amersham. Specifically, 1 μL of the undiluted QCMS reaction was mixed with 5 μL of sample buffer from the Templiphi kit. The mixture was heated for 3 minutes at 95° C. to denature the DNA. The reaction was placed on ice to cool for 2 minutes. Then, 5 μL of reaction buffer and 0.2 μL of phi29 polymerase were added to the reaction and incubated at 30° C. in a MJ Research thermocycler for 5 hours. Finally, the phi29 enzyme in the reaction was heat inactivated by incubation at 65° C. for 10 min in a MJ Research thermocycler. The amplified DNA was diluted 10-100 fold and 1 μL was used to transform 100 μL of competent B. subtilis BG6006 cells (Vogtentanz et al., 2007). Aliquots of 25 μL or 75 μL of the transformation mixture were plated on Luria Agar plates supplemented with 5 μg/ml chloramphenicol. After growth, individual transformants were picked to inoculate 200 μl of Luria broth supplemented with 5 μg/ml chloramphenicol in each well of a 96-well microtiter plate. The cultures were grown in a humidified and aerated box at 37° C. and 270 rpm (Innova 4230 incubator, New Brunswick Scientific). After growth, 80 μl of 50% glycerol were added to each well and mixed. A duplicate plate was made by removing 140 μl of the culture from each well and placing it in a new microtiter plate. Both plates were stored at −80° C. One plate was saved as a master and the other was submitted for DNA sequencing (Cogenics, Houston, Tex.) of the individual mutants. For DNA sequencing, an overnight culture of each colony grown in a microtiter plate was diluted 10-40 fold and 2 μL was used in a PCR reaction with Amersham Biosciences Ready-to-go PCR beads and the primers BBI-PCR-F and BBI-PCR-R. The thermo-cycling conditions were one cycle at 95° C. for 5 minutes, followed by 30 cycles of 95° C. for 1 minute, 55° C. for 1 minute and 72° C. for 1 minute, and a final cycle at 72° C. for 7 minutes. The PCR products were treated with Exonuclease I and shrimp alkaline phosphatase (EXOSAP-IT, Amersham Biosciences) or they were column purified before sequencing with the M13 reverse primer.

TABLE 1 NNS mutagenesis primers Primer name Primer Sequence SEQ ID NO: Forward Primers BBI1F GATCCCATACCGGATCCANNSGATGAG 196 AGCTCTAAACC BBI2F CCATACCGGATCCAGACNNSGAGAGCT 197 CTAAACCCTG BBI3F CATACCGGATCCAGACGATNNSAGCTC 198 TAAACCCTGTTG BBI4F CGGATCCAGACGATGAGNNSTCTAAAC 199 CTGTTGCGATC BBI5F GATCCAGACGATGAGAGCNNSAAACCC 200 TGTTGCGATCAATG BBI6F CAGACGATGAGAGCTCTNNSCCCTGTT 201 GCGATCAATG BBI7F GACGATGAGAGCTCTAAANNSTGTTGC 202 GATCAATGCGC BBI8F GATGAGAGCTCTAAACCCNNSTGCGAT 203 CAATGCGCATG BBI9F GAGAGCTCTAAACCCTGTNN 204 SGATCAATGCGCATGTAC BBI10F GCTCTAAACCCTGTTGCNNSCAATGCG 205 CATGTACGAAATC BBI11F CTAAACCCTGTTGCGATNNSTGCGCAT 206 GTACGAAATC BBI12F CTAAACCCTGTTGCGATCAANNSGCAT 207 GTACGAAATCAAATC BBI13F CCTGTTGCGATCAATGCNNSTGTACGA 208 AATCAAATCC BBI14F GTTGCGATCAATGCGCANNSACGAAAT 209 CAAATCCTCC BBI15F GCGATCAATGCGCATGTNNSAAATCAA 210 ATCCTCCACAG BBI16F GATCAATGCGCATGTACGNNSTCAAAT 211 CCTCCACAGTG BBI17F CAATGCGCATGTACGAAANNSAATCCT 212 CCACAGTGTCG BBI18F GCGCATGTACGAAATCANNSCCTCCAC 213 AGTGTCGGTG BBI19F CATGTACGAAATCAAATNNSCCACAGT 214 GTCGGTGTTC BBI20F GTACGAAATCAAATCCTNNSCAGTGTC 215 GGTGTTCCGATAT BBI21F CGAAATCAAATCCTCCANNSTGTCGGT 216 GTTCCGATATG BBI22F GAAATCAAATCCTCCACAGNNSCGGTG 217 TTCCGATATGCG BBI23F CAAATCCTCCACAGTGTNNSTGTTCCG 218 ATATGCGTCTG BBI24F CAAATCCTCCACAGTGTCGGNNSTCCG 219 ATATGCGTCTGAATTC BBI25F CTCCACAGTGTCGGTGTNNSGATATGC 220 GTCTGAATTC BBI26F CACAGTGTCGGTGTTCCNNSATGCGTC 221 TGAATTCCTG BBI27F CAGTGTCGGTGTTCCGATNNSCGTCTG 222 AATTCCTGTCATAG BBI28F GTCGGTGTTCCGATATGNNSCTGAATT 223 CCTGTCATAG BBI29F GGTGTTCCGATATGCGTNNSAATTCCT 224 GTCATAGTGC BBI30F GTTCCGATATGCGTCTGNNSTCCTGTC 225 ATAGTGCCTG BBI31F CCGATATGCGTCTGAATNNSTGTCATA 226 GTGCCTGCAAAAG BBI32F GATATGCGTCTGAATTCCNNSCATAGT 227 GCCTGCAAAAG BBI33F ATATGCGTCTGAATTCCTGTNNSAGTG 228 CCTGCAAAAGCTG BBI34F GTCTGAATTCCTGTCATNNSGCCTGCA 229 AAAGCTGCGC BBI35F CTGAATTCCTGTCATAGTNNSTGCAAA 230 AGCTGCGCATG BBI36F GAATTCCTGTCATAGTGCCNNSAAAAG 231 CTGCGCATGTTATAA BBI37F CCTGTCATAGTGCCTGCNNSAGCTGCG 232 CATGTTATAAC BBI38F GTCATAGTGCCTGCAAANNSTGCGCAT 233 GTTATAACCTG BBI39F CATAGTGCCTGCAAAAGCNNSGCATGT 234 TATAACCTGTAC BBI40F GTGCCTGCAAAAGCTGCNNSTGTTATA 235 ACCTGTACGG BBI41F CCTGCAAAAGCTGCGCANNSTATAACC 236 TGTACGGGTG BBI42F GCAAAAGCTGCGCATGTNNSAACCTGT 237 ACGGGTGGAC BBI43F CAAAAGCTGCGCATGTTATNNSCTGTA 238 CGGGTGGACCTG BBI44F GCTGCGCATGTTATAACNNSTACGGGT 239 GGACCTGTTTTTG BBI45F GCGCATGTTATAACCTGNNSGGGTGGA 240 CCTGTTTTTG BBI46F CATGTTATAACCTGTACNNSTGGACCT 241 GTTTTTGCGTC BBI47F GTTATAACCTGTACGGGNNSACCTGTT 242 TTTGCGTCGAC BBI48F GTTATAACCTGTACGGGTGGNNSTGTT 243 TTTGCGTCGACATC BBI49F ATAACCTGTACGGGTGGACCNNSTTTT 244 GCGTCGACATCAC BBI50F CTGTACGGGTGGACCTGTNNSTGCGTC 245 GACATCACGGAC BBI51F GTACGGGTGGACCTGTTTTNNSGTCGA 246 CATCACGGACTTC BBI52F GGTGGACCTGTTTTTGCNNSGACATCA 247 CGGACTTCTG BBI53F GGACCTGTTTTTGCGTCNNSATCACGG 248 ACTTCTGCTATG BBI54F CCTGTTTTTGCGTCGACNNSACGGACT 249 TCTGCTATGAAC BBI55F GTTTTTGCGTCGACATCNNSGACTTCT 250 GCTATGAACC BBI56F GTTTTTGCGTCGACATCACGNNSTTCT 251 GCTATGAACCATG BBI57F GCGTCGACATCACGGACNNSTGCTATG 252 AACCATGTAAAC BBI58F GTCGACATCACGGACTTCNNSTATGAA 253 CCATGTAAACC BBI59F GACATCACGGACTTCTGCNNSGAACCA 254 TGTAAACCAAG BBI60F CATCACGGACTTCTGCTATNNSCCATG 255 TAAACCAAGCGAG BBI61F CGGACTTCTGCTATGAANNSTGTAAAC 256 CAAGCGAGTAAAA BBI62F GACTTCTGCTATGAACCANNSAAACCA 257 AGCGAGTAAAAG BBI63F CTTCTGCTATGAACCATGTNNSCCAAG 258 CGAGTAAAAGCTTAA BBI64F GCTATGAACCATGTAAANNSAGCGAGT 259 AAAAGCTTAAC BBI65F CTATGAACCATGTAAACCANNSGAGTA 260 AAAGCTTAACTC BBI66F GAACCATGTAAACCAAGCNNSTAAAAG 261 CTTAACTCGAG Reverse Primers BBI1R GGTTTAGAGCTCTCATCSNNTGGATCC 262 GGTATGGGATC BBI2R CAGGGTTTAGAGCTCTCSNNGTCTGGA 263 TCCGGTATGG BBI3R CAACAGGGTTTAGAGCTSNNATCGTCT 264 GGATCCGGTATG BBI4R GATCGCAACAGGGTTTAGASNNCTCAT 265 CGTCTGGATCCG BBI5R CATTGATCGCAACAGGGTTTSNNGCTC 266 TCATCGTCTGGATC BBI6R CATTGATCGCAACAGGGSNNAGAGCTC 267 TCATCGTCTG BBI7R GCGCATTGATCGCAACASNNTTTAGAG 268 CTCTCATCGTC BBI8R CATGCGCATTGATCGCASNNGGGTTTA 269 GAGCTCTCATC BBI9R GTACATGCGCATTGATCSNNACAGGGT 270 TTAGAGCTCTC BBI10R GATTTCGTACATGCGCATTGSNNGCAA 271 CAGGGTTTAGAGC BBI11R GATTTCGTACATGCGCASNNATCGCAA 272 CAGGGTTTAG BBI12R GATTTGATTTCGTACATGCSNNTTGAT 273 CGCAACAGGGTTTAG BBI13R GGATTTGATTTCGTACASNNGCATTGA 274 TCGCAACAGG BBI14R GGAGGATTTGATTTCGTSNNTGCGCAT 275 TGATCGCAAC BBI15R CTGTGGAGGATTTGATTTSNNACATGC 276 GCATTGATCGC BBI16R CACTGTGGAGGATTTGASNNCGTACAT 277 GCGCATTGATC BBI17R CGACACTGTGGAGGATTSNNTTTCGTA 278 CATGCGCATTG BBI18R CACCGACACTGTGGAGGSNNTGATTTC 279 GTACATGCGC BBI19R GAACACCGACACTGTGGSNNATTTGAT 280 TTCGTACATG BBI20R ATATCGGAACACCGACACTGSNNAGGA 281 TTTGATTTCGTAC BBI21R CATATCGGAACACCGACASNNGGAGGA 282 TTTGATTTCG BBI22R CGCATATCGGAACACCGSNNCTGTGGA 283 GGATTTGATTTC BBI23R CAGACGCATATCGGAACASNNACACTG 284 TGGAGGATTTG BBI24R GAATTCAGACGCATATCGGASNNCCGA 285 CACTGTGGAGGATTTG BBI25R GAATTCAGACGCATATCSNNACACCGA 286 CACTGTGGAG BBI26R CAGGAATTCAGACGCATSNNGGAACAC 287 CGACACTGTG BBI27R CTATGACAGGAATTCAGACGSNNATCG 288 GAACACCGACACTG BBI28R CTATGACAGGAATTCAGSNNCATATCG 289 GAACACCGAC BBI29R GCACTATGACAGGAATTSNNACGCATA 290 TCGGAACACC BBI30R CAGGCACTATGACAGGASNNCAGACGC 291 ATATCGGAAC BBI31R CTTTTGCAGGCACTATGACASNNATTC 292 AGACGCATATCGG BBI32R CTTTTGCAGGCACTATGSNNGGAATTC 293 AGACGCATATC BBI33R CAGCTTTTGCAGGCACTSNNACAGGAA 294 TTCAGACGCATAT BBI34R GCGCAGCTTTTGCAGGCSNNATGACAG 295 GAATTCAGAC BBI35R CATGCGCAGCTTTTGCASNNACTATGA 296 CAGGAATTCAG BBI36R TTATAACATGCGCAGCTTTTSNNGGCA 297 CTATGACAGGAATTC BBI37R GTTATAACATGCGCAGCTSNNGCAGGC 298 ACTATGACAGG BBI38R CAGGTTATAACATGCGCASNNTTTGCA 299 GGCACTATGAC BBI39R GTACAGGTTATAACATGCSNNGCTTTT 300 GCAGGCACTATG BBI40R CCGTACAGGTTATAACASNNGCAGCTT 301 TTGCAGGCAC BBI41R CACCCGTACAGGTTATASNNTGCGCAG 302 CTTTTGCAGG BBI42R GTCCACCCGTACAGGTTSNNACATGCG 303 CAGCTTTTGC BBI43R CAGGTCCACCCGTACAGSNNATAACAT 304 GCGCAGCTTTTG BBI44R CAAAAACAGGTCCACCCGTASNNGTTA 305 TAACATGCGCAGC BBI45R CAAAAACAGGTCCACCCSNNCAGGTTA 306 TAACATGCGC BBI46R GACGCAAAAACAGGTCCASNNGTACAG 307 GTTATAACATG BBI47R GTCGACGCAAAAACAGGTSNNCCCGTA 308 CAGGTTATAAC BBI48R GATGTCGACGCAAAAACASNNCCACCC 309 GTACAGGTTATAAC BBI49R GTGATGTCGACGCAAAASNNGGTCCAC 310 CCGTACAGGTTAT BBI50R GTCCGTGATGTCGACGCASNNACAGGT 311 CCACCCGTACAG BBI51R GAAGTCCGTGATGTCGACSNNAAAACA 312 GGTCCACCCGTAC BBI52R CAGAAGTCCGTGATGTCSNNGCAAAAA 313 CAGGTCCACC BBI53R CATAGCAGAAGTCCGTGATSNNGACGC 314 AAAAACAGGTCC BBI54R GTTCATAGCAGAAGTCCGTSNNGTCGA 315 CGCAAAAACAGG BBI55R GGTTCATAGCAGAAGTCSNNGATGTCG 316 ACGCAAAAAC BBI56R CATGGTTCATAGCAGAASNNCGTGATG 317 TCGACGCAAAAAC BBI57R GTTTACATGGTTCATAGCASNNGTCCG 318 TGATGTCGACGC BBI58R GGTTTACATGGTTCATASNNGAAGTCC 319 GTGATGTCGAC BBI59R CTTGGTTTACATGGTTCSNNGCAGAAG 320 TCCGTGATGTC BBI60R CTCGCTTGGTTTACATGGSNNATAGCA 321 GAAGTCCGTGATG BBI61R TTTTACTCGCTTGGTTTACASNNTTCA 322 TAGCAGAAGTCCG BBI62R CTTTTACTCGCTTGGTTTSNNTGGTTC 323 ATAGCAGAAGTC BBI63R TTAAGCTTTTACTCGCTTGGSNNACAT 324 GGTTCATAGCAGAAG BBI64R GTTAAGCTTTTACTCGCTSNNTTTACA 325 TGGTTCATAGC BBI65R GAGTTAAGCTTTTACTCSNNTGGTTTA 326 CATGGTTCATAG BBI66R CTCGAGTTAAGCTTTTASNNGCTTGGT 327 TTACATGGTTC

The cultures in each master plate were thawed and a pin replicator was used to inoculate a new sterile microtiter plate containing 150 μl of Luria broth with 5 μg/ml chloramphenicol in each well. The plates were grown for ˜10 hrs in a humidified and aerated box as described above. For controls, the cultures in two wells were replaced with BBI and BBI-AV cultures that had been grown concurrently in 15 ml culture tubes (5 ml of Luria broth with 5 μg/ml chloramphenicol). After the cultures were grown, the plate was used to inoculate two duplicate 96-well filter plates (Millipore, MSGV2210) containing 150 μl of MBD medium (Vogtentanz et al., 2007) with 5 μg/ml chloramphenicol in each well by using a pin replicator. The duplicate plates were grown for ˜14 hours (as above, with plastic spacers placed between the stacked plates to allow for efficient air flow around the plates) and then 2-mecaptoethanol was added to each well in one of the plates to a final concentration of 2 mM. The plates were then grown for an additional ˜46 hours. The 96-well filter plates containing the culture broth (with or without added 2-mecaptoethanol) were then assayed for BBI and BCE activity.

Example 9 Primary Screening of BBIt-AV Variants BBI and BCE Activity Assays to Determine Variants Having the Highest BBIt-AV:BCE Activity Ratio

In this Example, methods are provided for assessing the effect of the amino acid substitutions introduced into the modified variant BBIt-AVs generated according to the site-saturation methods. The trypsin inhibitory activity of the modified BBIt-AV generated by the site-saturation methods described above was measured and compared to the trypsin inhibitory activity of the control wild-type BBI (SEQ ID NO:13; BBI-wt or sBBI) and the control unmodified BBIt-AV (SEQ ID NO:187). It is not intended that the present invention be limited to these specific methods, as other suitable methods find use.

Trypsin Inhibitory Assay: BBI Activity Determination

The relative concentration of active control and modified BBIt-AV's was determined by a trypsin inhibitory assay using purified sBBI as a standard as described above. For some samples, the active BBI concentration was measured by residual trypsin activity according to the method described by Flecker (Flecker, P. FEBS Lett. 252:153-157 [1989]; Vogtentanz et al. supra).

Libraries were analyzed using dilutions of cultures grown in 96-well plates. Typically, the culture broth from each well was diluted (200 fold) in Assay Buffer (100 mM Tris pH 8.6, 0.005% Tween 80) and transferred to a second microtiter plate (CoStar 9017, Corning, Inc., Corning, N.Y.). Then, 20 μl of the diluted samples were transferred to a third microtiter plate. A standard curve was created by adding 20, 10, 5 or 2.5 μl of the diluted sBBI (SEQ ID NO:13) control culture to empty wells. Then, 80 μL of 50 ng/ml trypsin (bovine pancreatic trypsin, Worthington Biochemical Corp., Lakewood, N.J.) (prepared from a 1 mg/mL stock solution diluted into Assay Buffer) was added to each well of the plate. The reactions were mixed and incubated 15 min at room temperature. After the incubation, 100 μL of 0.5 mg/ml trypsin substrate (succinyl-Ala-Ala-Pro-Arg-para-nitroanilide, Bachem Bioscience, Inc., King of Prussia, Pa.), diluted in Assay Buffer from a 100 mg/ml solution in DMSO, was added to each well, mixed, and the absorbance at 405 nm was monitored for 15 min, with 1 time point recorded every 12 sec using a Spectra Max 250 (Molecular Devices). The data points were used to determine the rate for each reaction. The standard curve was generated by plotting the reaction rate versus the BBI volume and was fitted to a four-parameter equation. All data fitting was done using software supplied by the manufacturer (Molecular Devices Corp., Sunnyvale, Calif.). The activity of each BBIt-AV variant was calculated from the standard curve. Thus, the activities of all modified variant proteins were determined relative to the activity in the sBBI control (BBI-WT) culture. If the majority of the data did not fall near the IC₅₀ of the standard curve, a new dilution plate was made and assayed.

Cellulase Enzyme Activity Assay: BCE Assay

The concentration of BCE was determined by an activity assay using purified BCE as a standard as described above and by (Vogtentanz et al. 2007). For the analysis of libraries grown in 96-well microtiter plates, 20 μl of culture broth were transferred to each well of a new microtiter plate. A standard curve was created by adding 20, 10, 5 or 2.5 μl of the diluted BBI-WT control culture to these empty wells. Then, 180 μl of the BCE substrate (0.25 μg/ml in Assay Buffer), 4-nitrophenyl β-D-cellobioside (Sigma, St. Louis, Mo.), was added to the plate. The plate was mixed and the absorbance at 405 nm monitored for 15 min, with 1 time point recorded every 12 s using a Spectra Max 250 (Molecular Devices Corp., Sunnyvale, Calif.). The data points were used to determine the rate for each reaction. The standard curve was generated by plotting rate versus culture volume and was fit to a quadratic curve. The BCE concentration in each variant culture was determined relative to the standard control culture.

Enzyme Activity Ratio: BBIt-AV:BCE Ratios

From the microtiter plate assay results, the BBIt-AV:BCE activity ratio of each well was determined by dividing the relative BBIt-AV trypsin inhibitory activity values by the relative BCE activity values. The data were sorted based on the BBIt-AV:BCE activity ratio. Modified variants with the highest BBIt-AV:BCE activity ratios were selected and subjected to a secondary screening described below.

Analysis of Enzyme Activity ratio BBIt-AV:BCE—Secondary Screening of Variants

In this example, modified BBIt-AVs having improved BBIt-AV:BCE activity relative to that of the control unmodified BBIt-AV when grown in the presence or absence of a reducing agent (2-mercaptoethanol), as determined in the primary screen, were arrayed and assayed in quadruplicate in a secondary screen. Initially, only the polynucleotides encoding each of the modified BBIt-AVs having the greatest BBIt-AV:BCE ratio as determined in the secondary screen were sequenced. Later, all BBIt-AV clones were sequenced.

Quadruplicate Testing in Microtiter Plates—Secondary Screen

For each plate, twenty-two samples of cultures containing modified BBIt-AVs selected from the initial screen, and two samples of control cultures containing sBBI and unmodified BBIt-AV, were grown for ˜10 hrs in 5 ml of Luria broth with 5 ppm chloramphenicol (37° C., 250 rpm) in 15 ml culture tubes. Then, 150 μl of each of the 24 cultures were arrayed in quadruplicate in a 96-well microtiter plate. This arrayed plate was then used to inoculate duplicate filter plates (Millipore, MSGV2210) containing 150 μl of MBD medium with 5 μg/ml chloramphenicol per well by using a pin replicator. The plated cultures were grown (in the presence and absence of 2-β-mercaptoethanol (βME), and the BBI-AV:BCE ratios were determined and analyzed as described above.

FIG. 10 shows an example of the effect of all possible 19 amino acid substitutions at position 29 of SEQ ID NO:187 on the trypsin inhibitory activity of the modified variant BBIt. The data show the ratio of BBI and BCE activities determined from quadruplicate measurements of BCE and trypsin activities of the modified BBI-AVs having a single amino acid substitution at position 29 of SEQ ID NO:187.

Example 10 Selection for Improved Modified BBIt-AVs with Known Amino Acid Substitutions

In this example, site saturation libraries were constructed to generate a complete set of amino acid substitutions at sites identified in the initial screen that resulted in BBIt-AVs having improved trypsin inhibitory activity when compared to the trypsin inhibitory activity of the unmodified parent BBI-AV. In addition, a complete set of amino acid substitutions at sites that had not been previously substituted with all possible 19 amino acids were generated by site-directed mutagenesis. A total of 39 site saturation libraries were generated and quadruplicate activity measurements of BBI trypsin inhibitory activity and BCE activity were analyzed to determine the single substitutions that resulted in modified BBIt-AV molecules having trypsin inhibitory activity that was greater than that of the control BBI molecules i.e. sBBI and unmodified BBIt-AV. The positions of the amino acids that were substituted in the unmodified BBIt-AV are shown in FIG. 9.

Construction of a Second Set of Site-Saturation Libraries at Sites 18, 38 and 61

First, to aid library construction, a synthetic gene was synthesized to replace the EcoRI site with a SphI site between the region coding for the trypsin inhibitory loop and the region encoding the chymotypsin inhibitory loop in the 2BBIck81 coding region (the location of the EcoRI site is shown in FIG. 3). The synthetic gene sequence is: 5′-GGATCCAGACGATGAGAGCTCTAAACCTTGTTGCGATCAATGCGCTTGTACAAAAT CAAACCCTCCACAATGTCGTTGTTCTGATATGCGTTTAAATAGCTGTCATTCTGCAT GCAAATCATGTGCTTGCTATAACCTTTACGGTTGGACATGTTTCTGCGTCGACATCA CTGACTTCTGCTATGAACCATGTAAACCTTCTGAATAAAAGCTT (SEQ ID NO:329). This synthetic gene was cloned into p2JMagk103-Ink2-BBIt-AV as a BamHI-HindIII fragment and the resulting vector was called p2JMagk103-Ink2-BBIt-AVsph.

Site-saturation libraries were constructed at positions 18, 37 and 61 using oligonucleotide cassettes as follows.

A site-saturation library at site 18 was constructed by annealing oligonucleotides 5′-GTACAAAATCANNSCCTCCACAATGTCGTTGTTCTGATATGCGTTTAAATAGCTGTCATTCTGCATG (SEQ ID NO:330) and 5′-CAGAATGACAGCTATTTAAACGCATATCAGAACAACGACATTGTGGAGGSNNTGATTTT (SEQ ID NO:331) and ligating the resulting cassette into the BsrGI and SphI sites of p2JMagk103-Ink2-BBI-AVsph.

A site-saturation library at site 37 was constructed by annealing the oligonucleotides 5′-TCGACGCAGAAACATGTCCAACCGTAAAGGTTATAGCAAGCACATGASNNGCATG (SEQ ID NO:332) and 5′-CNNSTCATGTGCTTGCTATAACCTTTACGGTTGGACATGTTTCTGCG (SEQ ID NO:333) and ligating the resulting cassette into the SalI and SphI sites of p2JMagk103-Ink2-BBI-AVsph.

The site-saturation library at position 61 was made by annealing oligonucleotide 5′-TCGACATCACTGACTTCTGCTATGAANNSTGTAAACCTTCTGAATAAA (SEQ ID NO:334) with the two oligonucleotides 5′-TTCATAGCAGAAGTCAGTGATG (SEQ ID NO:335) and 5′-AGCTTTTATTCAGAAGGTTTACA (SEQ ID NO:336) and ligating this cassette into the SalI and HindIII sites of p2JMagk103-Ink2-BBIt-AVsph.

The ligation mixes were used to transform E. coli TOP10 cells (Invitrogen). Ninety six transformants were selected, and the amino acid substitution present in each clone was determined by DNA sequencing. Plasmids isolated from each single amino acid substitution were then used to transform B. subtilis.

Generation of Amino Acid Substitutions by Site Directed Mutagenesis

Amino acid substitutions at selected sites were constructed by ligating an oligonucleotide or an oligonucleotide cassette into the of p2JMagk103-Ink2-BBI-AVsph vector using the appropriate restriction sites. The substitutions, oligonucleotide sequences and restriction sites used are shown in Table 2.

TABLE 2 Oligonucleotides for site directed muatagenesis Amino Acid Substitution(s) Oligonucleotide Pairs Sequence(s) Restriction Sites A13Y 5′-Phos-GTACAGTAGCATTGATCGCAACAAGGTT SacI & BsrGI TAGAGCT (SEQ ID NO:337) R23D, E 5′-Phos-GTACAAAATCAAACCCTCCACAATGTGA BsrGI & SphI NTGTTCTGATATGCGTTTAAATAGCTGTCATTCTGC ATG (SEQ ID NO:338) 5′-CAGAATGACAGCTATTTAAACGCATATCAGAAC ANTCACATTGTGGAGGGTTTGATTTT (SEQ ID NO:339) S25M 5′-Phos-GTACAAAATCAAACCCTCCACAATGTCG BsrGI & SphI TTGTATGGATATGCGTTTAAATAGCTGTCATTCTGC ATG (SEQ ID NO:340) 5′-CAGAATGACAGCTATTTAAACGCATATCCATAC AACGACATTGTGGAGGGTTTGATTTT (SEQ ID NO:341) S25W 5′-Phos-GTACAAAATCAAACCCTCCACAATGTCG BsrGI & SphI TTGTTGGGATATGCGTTTAAATAGCTGTCATTCTGC ATG (SEQ ID NO:342) 5′-CAGAATGACAGCTATTTAAACGCATATCCCAAC AACGACATTGTGGAGGGTTTGATTTT (SEQ ID NO:343) S25A, V, G 5′-Phos-GTACAAAATCAAACCCTCCACAATGTCG BsrGI & SphI TTGTGBAGATATGCGTTTAAATAGCTGTCATTCTGC ATG (SEQ ID NO:344) 5′-CAGAATGACAGCTATTTAAACGCATATCTVCAC AACGACATTGTGGAGGGTTTGATTTT (SEQ ID NO:345) S25N, Q, K, H 5′-Phos-GTACAAAATCAAACCCTCCACAATGTCG BsrGI & SphI TTGTMAWGATATGCGTTTAAATAGCTGTCATTCTGC ATG (SEQ ID NO:346) 5′-CAGAATGACAGCTATTTAAACGCATATCWTKAC AACGACATTGTGGAGGGTTTGATTTT (SEQ ID NO:347) M27W 5′-Phos-GTACAAAATCAAACCCTCCACAATGTCG BsrGI & SphI TTGTTCTGATTGGCGTTTAAATAGCTGTCATTCTGC ATG (SEQ ID NO:348) 5′-CAGAATGACAGCTATTTAAACGCCAATCAGAAC AACGACATTGTGGAGGGTTTGATTTT (SEQ ID NO:349) M27H 5′-Phos-GTACAAAATCAAACCCTCCACAATGTCG BsrGI & SphI TTGTTCTGATCACCGTTTAAATAGCTGTCATTCTGC ATG (SEQ ID NO:350) 5′-CAGAATGACAGCTATTTAAACGGTGATCAGAAC AACGACATTGTGGAGGGTTTGATTTT (SEQ ID NO:351) M27G, A, V, D 5′-Phos-GTACAAAATCAAACCCTCCACAATGTCG BsrGI & SphI TTGTTCTGATGNTCGTTTAAATAGCTGTCATTCTGC ATG (SEQ ID NO:352) 5′-CAGAATGACAGCTATTTAAACGANCATCAGAAC AACGACATTGTGGAGGGTTTGATTTT (SEQ ID NO:353) M27F, I 5′-Phos-GTACAAAATCAAACCCTCCACAATGTCG BsrGI & SphI TTGTTCTGATWTTCGTTTAAATAGCTGTCATTCTGC ATG (SEQ ID NO:354) 5′-CAGAATGACAGCTATTTAAACGAAWATCAGAAC AACGACATTGTGGAGGGTTTGATTTT (SEQ ID NO:355) L29I 5′-Phos-GTACAAAATCAAACCCTCCACAATGTCG BsrGI & SphI TTGTTCTGATATGCGTATTAATAGCTGTCATTCTGC ATG (SEQ ID NO:356) 5′-CAGAATGACAGCTATTAATACGCATATCAGAAC AACGACATTGTGGAGGGTTTGATTTT (SEQ ID NO:357) L29D, E 5′-Phos-GTACAAAATCAAACCCTCCACAATGTCG BsrGI & SphI TTGTTCTGATATGCGTGANAATAGCTGTCATTCTGC ATG (SEQ ID NO:358) 5′-CAGAATGACAGCTATTNTCACGCATATCAGAAC AACGACATTGTGGAGGGTTTGATTTT (SEQ ID NO:359) S31M 5′-Phos-GTACAAAATCAAACCCTCCACAATGTCG BsrGI & SphI TTGTTCTGATATGCGTTTAAATATGTGTCATTCTGC ATG (SEQ ID NO:360) 5′-CAGAATGACACATATTTAAACGCATATCAGAAC AACGACATTGTGGAGGGTTTGATTTT (SEQ ID NO:361) S31D, E, Y 5′-Phos-GTACAAAATCAAACCCTCCACAATGTCG BsrGI & SphI TTGTTCTGATATGCGTTTAAATKAWTGTCATTCTGC ATG (SEQ ID NO:362) 5′-CAGAATGACAWTMATTTAAACGCATATCAGAAC AACGACATTGTGGAGGGTTTGATTTT (SEQ ID NO:363) S31Q, H 5′-Phos-GTACAAAATCAAACCCTCCACAATGTCG BsrGI & SphI TTGTTCTGATATGCGTTTAAATCANTGTCATTCTGC ATG (SEQ ID NO:365) 5′-CAGAATGACANTGATTTAAACGCATATCAGAAC AACGACATTGTGGAGGGTTTGATTTT (SEQ ID NO:365) S34I 5′-Phos-GTACAAAATCAAACCCTCCACAATGTCG BsrGI & SphI TTGTTCTGATATGCGTTTAAATAGCTGTCATATCGC ATG (SEQ ID NO:366) 5′-CGATATGACAGCTATTTAAACGCATATCAGAAC AACGACATTGTGGAGGGTTTGATTTT (SEQ ID NO:367) S34W 5′-Phos-GTACAAAATCAAACCCTCCACAATGTCG BsrGI & SphI TTGTTCTGATATGCGTTTAAATAGCTGTCATTGGGC ATG (SEQ ID NO:368) 5′-CCCAATGACAGCTATTTAAACGCATATCAGAAC AACGACATTGTGGAGGGTTTGATTTT (SEQ ID NO:369) S34R, Q 5′-Phos-GTACAAAATCAAACCCTCCACAATGTCG BsrGI & SphI TTGTTCTGATATGCGTTTAAATAGCTGTCATCRAGC ATG (SEQ ID NO:370) 5′-CTYGATGACAGCTATTTAAACGCATATCAGAAC AACGACATTGTGGAGGGTTTGATTTT (SEQ ID NO:371) S34D, E 5′-Phos-GTACAAAATCAAACCCTCCACAATGTCG BsrGI & SphI TTGTTCTGATATGCGTTTAAATAGCTGTCATGANGC ATG (SEQ ID NO:372) 5′-CNTCATGACAGCTATTTAAACGCATATCAGAAC AACGACATTGTGGAGGGTTTGATTTT (SEQ ID NO:373) A40V, D 5′-Phos-TCGACGCAGAAACATGTCCAACCGTAAA SphI & SalI GGTTATAGCAAWCACATGATTTGCATG (SEQ ID NO:374) 5′-CAAATCATGTGWTTGCTATAACCTTTACGGTTG GACATGTTTCTGCG (SEQ ID NO:375) A40H, P 5′-Phos-TCGACGCAGAAACATGTCCAACCGTAAA SphI & SalI GGTTATAGCAAKGACATGATTTGCATG (SEQ ID NO:376) 5′-CAAATCATGTCMTTGCTATAACCTTTACGGTTG GACATGTTTCTGCG (SEQ ID NO:377) A40Y, W 5′-Phos-TCGACGCAGAAACATGTCCAACCGTAAA SphI & SalI GGTTATAGCAMYAACATGATTTGCATG (SEQ ID NO:378) 5′-CAAATCATGTTRKTGCTATAACCTTTACGGTTG GACATGTTTCTGCG F50D 5′-Phos-TCGACGCAATCACATGTCCAACCGTAAA SphI & SalI GGTTATAGCAAGCACATGATTTGCATG (SEQ ID NO:380) 5′-CAAATCATGTGCTTGCTATAACCTTTACGGTTG GACATGTGATTGCG (SEQ ID NO:381) F50Q, P 5′-Phos-TCGACGCATKGACATGTCCAACCGTAAAG SphI & SalI GTTATAGCAAGCACATGATTTGCATG (SEQ ID NO:382) 5′-CAAATCATGTGCTTGCTATAACCTTTACGGTTG GACATGTCMATGCG (SEQ ID NO:383) F50S, R 5′-Phos-TCGACGCAKCTACATGTCCAACCGTAAA SphI & SalI GGTTATAGCAAGCACATGATTTGCATG (SEQ ID NO:384) 5′-CAAATCATGTGCTTGCTATAACCTTTACGGTTG GACATGTAGMTGCG (SEQ ID NO:385) V52D 5′-Phos-CAAATCATGTGCTTGCTATAACCTTTAC SphI & HindIII GGGTGGACATGTTTCTGCGATGACATCACTGACTTCT GCTATGAACCATGTAAACCTTCTGAATAAA (SEQ ID NO:386) 5′-AGCTTTTATTCAGAAGGTTTACATGGTTCATAG CAGAAGTCAGTGATGTCATCGCAGAAACATGTCCAA CCGTAAAGGTTATAGCAAGCACATGA TTTGCATG (SEQ ID NO:387)

Secondary Screening of Individual Amino Acid Substitutions: Quadruplicate Testing in Microtiter Plates

For each chosen site, clones were selected corresponding to individual amino acid substitutions. These clones were grown, along with cultures of BBI-WT, BBIt-AV and BBIt-AV-F50T as controls, in 5 ml of Luria broth with 5 ppm chloramphenicol (37° C., 250 rpm) in 15 ml culture tubes for ˜10 hrs at 37° C. One hundred and fifty microliter aliquots of each the cultures were arrayed in the wells of a 96-well microtiter plate, 4 wells for each amino acid substitution and each control. Each culture grown in the arrayed plate was then used to inoculate duplicate filter plates (Millipore, MSGV2210) containing 150 μl of MBD medium with 5 μg/ml chloramphenicol per well by using a pin replicator. The cultures in the plates were grown in the presence and absence of 2-mercaptoethanol and analyzed as described above. BBIt-AV-F50T is a modified BBIt-AV that was identified as having an initial BBIt-AV:BCE activity ratio prior to activation that was comparable to that of the unmodified BBIt-AV parent, but which has a three-fold greater BBIt-AV:BCE activity ratio than that of the unmodified BBIt-AV after activation with a reducing agent such as 2-mercaptoethanol.

For each site, the BBI:BCE activity ratio was determined for each well and the average values for the quadruplicate measurements was calculated. The activity ratios for the individual amino acid substitutions were compared to the ratios calculated for unmodified parent BBIt-AV (SEQ ID NO:187), the wild-type BBI (SEQ ID NO:13) and the modified BBIt-AV named BBIt-AV-F50T.

FIGS. 11A and 11B show the average of the quadruplicate determinations of the BBI:BCE ratio determined in the presence of 2-mercaptoethanol (BME) relative to that determined for the sBBI for each of the substitutions at positions 50 and 37, respectively.

Overall, the data show single amino acid substitutions made at 16 of the 66 positions/sites in the parent BBIt-AV molecule resulted in BBIt-AV variants that had trypsin inhibitory activity equal or greater than that of the precursor BBIt-AV (data summarized in Table 3). Of all the single site substitutions, F50R resulted in the best BBIt-AV:BCE− activity ratio, while several substitutions at position 50 produced modified variant BBIt-AVs having a four-fold greater BBIt-AV:BCE activity ratios than that of the precursor unmodified BBIt-AV, and that were comparable to that of the wild-type inhibitor (FIG. 11A-B).

These data show that a single substitution in the BBI-AV molecule can have a significant effect on the activity of the BBI-AV.

TABLE 3 Substitutions that result in modified BBIt-AV molecules with greater BBI:BCE activity ratios than the unmodified BBIt-AV Position Native Amino Acid Improved Substitutions (with BME) 1 D A, P, 4 S V (4D13 insert)* 5 S P, A 11 Q G 13 A Y, I, F, M, L V, K, R 18 N I, V 25 S K N, W, I, A, R 27 M H, R, K, V, A, Q 29 L R, K, P 31 S Q, H, E, A, R, W, K, T 38 S N, K, R 40 A H, K, Q, R, Y 50 F R, Q, K, T, V, M, S 52 V K, T, R, Q, L, H, A, M, S, E 55 T M 65 S E D *The 4D13 insert is a 13 amino acid sequence that was added to the N-terminus of the BBI protein as described below.

Example 11 Selection of Modified Variants Having Improved Purification Yield

In this example, the methods described were used to identify modified BBIt-AVs containing single amino acid substitutions that retained trypsin inhibitory activity while being produced at yields greater than the corresponding unmodified precursor BBIt-AV.

Modified BBI-AVs that had been selected for having the greatest BBIt-AV:BCE activity ratio in the quadruplicate plate screens were further tested in a shake flask screen that was designed to mimic the acid/heat treatment that is used during the purification process to cleave the BBIt-AV from the BCE:BBIt-AV fusion protein. Thus, modified BBIt-AVs that retained a BBI:BCE activity ratio greater than that of the unmodified BBI-AV following acid/heat treatment were identified as modified BBIt-AVs that would be produced at yields greater than that of the unmodified precursor BBIt-AV.

After activation with reducing agent, e.g. 2-mercaptoethanol, a higher BBIt-AV:BCE activity ratio indicates that a given amino acid substitution has significantly increased the fraction of molecules with at least a correctly folded trypsin inhibitory loop. However, two different amino acid substitutions with similar BBIt-AV:BCE activity ratios could have somewhat different total yields after purification. For example, V52L and M27A activate to similar levels but M27A has about 40% better yield after acid/heat treatment. Thus, variants selected in the first screens as “good activators” were further evaluated by a second criterion that was developed to better predict purification yield.

Shake Flask Screen: Testing for the Purification Yield

To increase expression, the gene copy of the selected clones was amplified by sequentially streaking colonies on Luria agar plates with 25 μg/ml chloramphenicol until the growth rate was similar to growth on Luria agar plates without chloramphenicol. Cultures of the selected variants and controls were grown in 3 ml Luria broth with 25 μg/ml chloramphenicol in 15 ml tubes for ˜10 h. These cultures (30 μl) were then used to inoculate 30 ml of MBD medium in 250 ml baffled shake flasks and grown at 37° C. and 225 rpm for ˜60 h.

For selection of the variants with the best yields, the culture broths were processed as follows. The broth was first activated by mixing 20 ml of broth with 20 ml of 0.25 M glycine buffer (pH 9.3) and the pH was adjusted to 9.0 with 50% NaOH. Then, 2-mercaptoethanol was added to the diluted cultures to a final concentration of 2 mM and the cultures were incubated overnight at room temperature with gentle shaking. The BBIt-AV moiety was then cleaved from the fusion protein by acid/heat treatment, which was accomplished by first adjusting the pH of the activated broth to pH 1.9-2.0 with sulfuric acid, and followed by incubation at 60° C. for 16 hours, with shaking. The BBIt-AV-BCE fusion protein and the BCE catalytic domain are not soluble at pH 2, whereas the free BBI species is soluble. After the cleavage reaction, the insoluble material was removed by centrifugation (5 min. at 3,000 rpm) and the supernatant was analyzed for trypsin inhibitory activity as described above. The BBI activity was also determined for the starting material and for the samples taken after activation. The BCE activity of the starting material was also determined as described above. The BBIt-AV:BCE activity ratios were then calculated for each step in process. The modified BBIt-AVs produced at the highest yields following the acid/heat treatment were selected for further study.

Amino acid substitutions were found at sites 13, 25, 27, 29, 31, 40, 50 and 52 that significantly improved the BBIt-AV yield after acid/heat treatment. Other substitutions with improved yields after acid/heat treatment were D1C (when not activated), S4V, S5P, Q11G, S38N and S65E. An additional variant that was produced by an unexpected duplication of a primer (during the QuikChange reaction used to make the site-saturation library at position 4) also had a higher BBIt-AV:BCE activity ratio after acid/heat treatment. In this variant, the amino acid sequence, DDEPSKPCCDPDP (SEQ ID NO:389) (called the 4D13 insert), was inserted between the linker and the N-terminus of BBIt-AV (SEQ ID NO:187) to generate a modified BBIt-AV fused to the linker in SEQ ID NO:391.

(SEQ ID NO:391) DNNDPIPDPddepskpccdpdpDDESSKPCCDQCACTKSNPPQCRCSDMR LNSCHSACKSCACYNLYGWTCFCVDITDFCYEPCKPSE.

The linker is shown in italicized letters, the insert is written in lower case letters and the BBIt-AV corresponding to SEQ ID NO:187 is in bold letters.

The sequence of the 4D13 modified BBtl-AV is:

(SEQ ID NO:390) DDEPSKPCCDPDPDDESSKPCCDQCACTKSNPPQCRCSDMRLNSCHSACK SCACYNLYGWTCFCVDITDFCYEPCKPSE.

The synthetic gene encoding 4D13-BBIt-AV of SEQ ID NO:390 is:

(SEQ ID NO:394) GGATCCAGATGACGAACCGAGCAAACCTTGCTGTGATCCAGACCCTGACG ATGAGAGCTCTAAACCCTGTTGCGATCAATGCATTTGTACGAAATCAAAT CCTCCACAGTGTCGGTGTTCCGATATGCGTCCGAATTCCTGTCATAGTGC CTGCAAAAGCTGCAAGTGTTATAACCTGTACGGGTGGACCTGTACATGCG CCGACATCACGGACTTCTGCTATGAACCATGTAAACCAAGCGAGTAAAAG CTT.

Example 12 Combinations of Mutations

In this example, the single amino acid substitutions that resulted in BBIt-AVs having improved BBI:BCE activity ratios in either the plate screens or in the shake flask screen were combined to generate modified BBIt-AVs that have greater BBIt-AV:BCE activity ratios and/or production yields than those of modified BBIt-AVs carrying single amino acid substitutions.

Following the primary and secondary screens described in Examples 9 and 10 above, three modified BBIt-AVs each containing a single amino acid substitution A13I, F50T and V52A, respectively, were identified as having an improved BBI:BCE activity ratio relative to the respective control. A library was constructed to select for the best combination of these three substitutions. The library was made by the QuikChange® mutagenesis protocol described above using equimolar concentrations of the following primers in the reaction mixture: primer 5′-CTGTTGCGATCAATGCATTTGTACGAAATC (SEQ ID NO:395) was used to generate the A13I substitution, primer 5′-CTGTACGGGTGGACCTGTACATGCGYCGACATCACGGACTTC (SEQ ID NO:396) was used to generate the F50T and F50T-V52A substitutions, and primer 5′-GACCTGTTTTTGCGYCGACATCACGGAC (SEQ ID NO:397) was used to generate the V52A substitution in the reaction mixture. Clones were selected and screened in microtiter plates and screened as described above.

The modified double variant BBIt-AV containing the F50T and the V52A substitutions (BBIt-AV-F50T-V52A; SEQ ID NO:595) was obtained, and determined to have the polynucleotide sequence of SEQ ID NO:398.

BBIt-AV-F50T-V52A (SEQ ID NO:398) GGATCCAGACGATGAGAGCTCTAAACCCTGTTGCGATCAATGCGCATGTA CGAAATCAAATCCTCCACAGTGTCGGTGTTCCGATATGCGTCTGAATTCC TGTCATAGTGCCTGCAAAAGCTGCGCATGTTATAACCTGTACGGGTGGAC CTGTACATGCGCCGACATCACGGACTTCTGCTATGAACCATGTAAACCAA GCGAGTAAAAGCTT (SEQ ID NO:595) DPDDESSKPCCDQCACTKSNPPQCRCSDMRLNSCHSACKSCACYNLYGWT CTCADITDFCYEPCKPSE.

Generation of Modified BBIt-AVs Containing Three, Four and Five Amino Acid Substitutions

Combinations of four productive substitutions, A13I, S25L, L29P and A40K, identified in the screens described in Examples 9 and 10 above were made in the BBIt-AV-F50T-V52A double variant. These substitutions were combined by using synthetic genes that were cloned into the BamHI and HindIII sites of the p2JMagk103Ink2BBIt-AV vector. The polynucleotide sequences encoding the corresponding amino acid sequences for the modified BBIt-AVs comprising combinations of substitutions are given below.

DDIt-AV-A13I-S25L-F50T-V52A (SEQ ID NO:399) GGATCCAGACGATGAGAGCTCTAAACCCTGTTGCGATCAATGCATTTGTA CGAAATCAAATCCTCCACAGTGTCGGTGTCTTGATATGCGTCTGAATTCC TGTCATAGTGCCTGCAAAAGCTGCGCATGTTATAACCTGTACGGGTGGAC CTGTACATGCGCCGACATCACGGACTTCTGCTATGAACCATGTAAACCAA GCGAGTAAAAGCTT (SEQ ID NO:596) DPDDESSKPCCDQCICTKSNPPQCRCLDMRLNSCHSACKSCACYNLYGWT CTCADITDFCYEPCKPSE BBIt-AV-A13I-S25L-L29P-F50T-V52A (SEQ ID NO:400) GGATCCAGACGATGAGAGCTCTAAACCCTGTTGCGATCAATGCATTTGTA CGAAATCAAATCCTCCACAGTGTCGGTGTCTTGATATGCGTCCGAATTCC TGTCATAGTGCCTGCAAAAGCTGCGCATGTTATAACCTGTACGGGTGGAC CTGTACATGCGCCGACATCACGGACTTCTGCTATGAACCATGTAAACCAA GCGAGTAAAAGCTT (SEQ ID NO:597) DPDDESSKPCCDQCICTKSNPPQCRCLDMRPNSCHSACKSCACYNLYGWT CTCADITDFCYEPCKPSE. BBIt-AV-A13I-S25L-L29P-A40K-F50T-V52A (SEQ ID NO:401) GGATCCAGACGATGAGAGCTCTAAACCCTGTTGCGATCAATGCATTTGTA CGAAATCAAATCCTCCACAGTGTCGGTGTCTTGATATGCGTCCGAATTCC TGTCATAGTGCCTGCAAAAGCTGCAAGTGTTATAACCTGTACGGGTGGAC CTGTACATGCGCCGACATCACGGACTTCTGCTATGAACCATGTAAACCAA GCGAGTAAAAGCTT (SEQ ID NO:598) DPDDESSKPCCDQCICTKSNPPQCRCLDMRPNSCHSACKSCKCYNLYGWT CTCADITDFCYEPCKPSE BBIt-AV-A13I-S25L-A40K-F50T-V52A (SEQ ID NO:402) GGATCCAGACGATGAGAGCTCTAAACCCTGTTGCGATCAATGCATTTGTA CGAAATCAAATCCTCCACAGTGTCGGTGTCTTGATATGCGTCTGAATTCC TGTCATAGTGCCTGCAAAAGCTGCAAGTGTTATAACCTGTACGGGTGGAC CTGTACATGCGCCGACATCACGGACTTCTGCTATGAACCATGTAAACCAA GCGAGTAAAAGCTT (SEQ ID NO:599) DPDDESSKPCCDQCICTKSNPPQCRCLDMRLNSCHSACKSCKCYNLYGWT CTCADITDFCYEPCKPSE BBIt-AV-A13I-L29P-F50T-V52A (SEQ ID NO:403) GGATCCAGACGATGAGAGCTCTAAACCCTGTTGCGATCAATGCATTTGTA CGAAATCAAATCCTCCACAGTGTCGGTGTTCCGATATGCGTCCGAATTCC TGTCATAGTGCCTGCAAAAGCTGCGCATGTTATAACCTGTACGGGTGGAC CTGTACATGCGCCGACATCACGGACTTCTGCTATGAACCATGTAAACCAA GCGAGTAAAAGCTT (SEQ ID NO:600) DPDDESSKPCCDQCICTKSNPPQCRCSDMRPNSCHSACASCACYNLYGWT CTCADITDFCYEPCKPSE BBIt-AV-A13I-L29P-A40K-F50T-V52A (TA5) (SEQ ID NO:404) GGATCCAGACGATGAGAGCTCTAAACCCTGTTGCGATCAATGCATTTGTA CGAAATCAAATCCTCCACAGTGTCGGTGTTCCGATATGCGTCCGAATTCC TGTCATAGTGCCTGCAAAAGCTGCAAGTGTTATAACCTGTACGGGTGGAC CTGTACATGCGCCGACATCACGGACTTCTGCTATGAACCATGTAAACCAA GCGAGTAAAAGCTT (SEQ ID NO:601) DPDDESSKPCCDQCICTKSNPPQCRCSDMRPNSCHSACKSCKCYNLYGWT CTCADITDFCYEPCKPSE BBIt-AV-A13I-A40K-F50T-V52A (SEQ ID NO:405) GGATCCAGACGATGAGAGCTCTAAACCCTGTTGCGATCAATGCATTTGTA CGAAATCAAATCCTCCACAGTGTCGGTGTTCCGATATGCGTCTGAATTCC TGTCATAGTGCCTGCAAAAGCTGCAAGTGTTATAACCTGTACGGGTGGAC CTGTACATGCGCCGACATCACGGACTTCTGCTATGAACCATGTAAACCAA GCGAGTAAAAGCTT (SEQ ID NO:602) DPDDESSKPCCDQCICTKSNPPQCRCSDMRLNSCHSACKSCKCYNLYGWT CTCADITDFCYEPCKPSE BBIt-AV-S25L-F50T-V52A (SEQ ID NO:406) GGATCCAGACGATGAGAGCTCTAAACCCTGTTGCGATCAATGCGCATGTA CGAAATCAAATCCTCCACAGTGTCGGTGTCTTGATATGCGTCTGAATTCC TGTCATAGTGCCTGCAAAAGCTGCGCATGTTATAACCTGTACGGGTGGAC CTGTACATGCGCCGACATCACGGACTTCTGCTATGAACCATGTAAACCAA GCGAGTAAAAGCTT (SEQ ID NO:603) DPDDESSKPCCDQCACTKSNPPQCRCLDMRLNSCHSACKSCACYNLYGWT CTCADITDFCYEPCKPSE BBIt-AV-S25L-L29P-F50T-V52A (SEQ ID NO:407) GGATCCAGACGATGAGAGCTCTAAACCCTGTTGCGATCAATGCGCATGTA CGAAATCAAATCCTCCACAGTGTCGGTGTCTTGATATGCGTCCGAATTCC TGTCATAGTGCCTGCAAAAGCTGCGCATGTTATAACCTGTACGGGTGGAC CTGTACATGCGCCGACATCACGGACTTCTGCTATGAACCATGTAAACCAA GCGAGTAAAAGCTT (SEQ ID NO:604) DPDDESSKPCCDQCACTKSNPPQCRCLDMRPNSCHSACKSCACYNLYGWT CTCADITDFCYEPCKPSE BBIt-AV-S25L-L29P-A40K-F50T-V52A (SEQ ID NO:408) GGATCCAGACGATGAGAGCTCTAAACCCTGTTGCGATCAATGCGCATGTA CGAAATCAAATCCTCCACAGTGTCGGTGTCTTGATATGCGTCCGAATTCC TGTCATAGTGCCTGCAAAAGCTGCAAGTGTTATAACCTGTACGGGTGGAC CTGTACATGCGCCGACATCACGGACTTCTGCTATGAACCATGTAAACCAA GCGAGTAAAAGCTT (SEQ ID NO:605) DPDDESSKPCCDQCACTKSNPPQCRCLDMRPNSCHSACKSCKCYNLYGWT CTCADITDFCYEPCKPSE BBIt-AV-S25L-A40K-F50T-V52A (SEQ ID NO:409) GGATCCAGACGATGAGAGCTCTAAACCCTGTTGCGATCAATGCGCATGTA CGAAATCAAATCCTCCACAGTGTCGGTGTCTTGATATGCGTCTGAATTCC TGTCATAGTGCCTGCAAAAGCTGCAAGTGTTATAACCTGTACGGGTGGAC CTGTACATGCGCCGACATCACGGACTTCTGCTATGAACCATGTAAACCAA GCGAGTAAAAGCTT (SEQ ID NO:606) DPDDESSKPCCDQCACTKSNPPQCRCLDMRLNSCHSACKSCKCYNLYGWT CTCADITDFCYEPCKPSE BBIt-AV-L29P-F50T-V52A (SEQ ID NO:410) GGATCCAGACGATGAGAGCTCTAAACCCTGTTGCGATCAATGCGCATGTA CGAAATCAAATCCTCCACAGTGTCGGTGTTCCGATATGCGTCCGAATTCC TGTCATAGTGCCTGCAAAAGCTGCGCATGTTATAACCTGTACGGGTGGAC CTGTACATGCGCCGACATCACGGACTTCTGCTATGAACCATGTAAACCAA GCGAGTAAAAGCTT (SEQ ID NO:607) DPDDESSKPCCDQCACTKSNPPQCRCSDMRPNSCHSACKSCACYNLYGWT CTCADITDFCYEPCKPSE BBIt-AV-L29P-A40K-F50T-V52A (SEQ ID NO:411) GGATCCAGACGATGAGAGCTCTAAACCCTGTTGCGATCAATGCGCATGTA CGAAATCAAATCCTCCACAGTGTCGGTGTTCCGATATGCGTCCGAATTCC TGTCATAGTGCCTGCAAAAGCTGCAAGTGTTATAACCTGTACGGGTGGAC CTGTACATGCGCCGACATCACGGACTTCTGCTATGAACCATGTAAACCAA GCGAGTAAAAGCTT (SEQ ID NO:608) DPDDESSKPCCDQCACTKSNPPQCRCSDMRPNSCHSACKSCKCYNLYGWT CTCADITDFCYEPCKPSE BBIt-AV-A40K-F50T-V52A (SEQ ID NO:412) GGATCCAGACGATGAGAGCTCTAAACCCTGTTGCGATCAATGCGCATGTA CGAAATCAAATCCTCCACAGTGTCGGTGTTCCGATATGCGTCTGAATTCC TGTCATAGTGCCTGCAAAAGCTGCAAGTGTTATAACCTGTACGGGTGGAC CTGTACATGCGCCGACATCACGGACTTCTGCTATGAACCATGTAAACCAA GCGAGTAAAAGCTT (SEQ ID NO:609) DPDDESSKPCCDQCACTKSNPPQCRCSDMRNLSCHSACKSCKCYNLYGWT CTCADITDFCYEPCKPSE

Generation of Modified BBIt-AVs Containing Six and Seven Amino Acid Substitutions

Additional modified BBIt-AV were generated to comprise variations including the 4D13 insertion, single amino acid substitutions D1C, S4V, S5P, Q11G, I13L, S25R, M27R, P29K, S31A, S31R, S38N, T50K, A52T, S65E, and double amino acid substitutions including S25R-S31R and S25R-S31 with substitutions present in a quintuple variant (BBIt-AV-A13I-L29P-A40K-F50T-V52A) encoded by the polynucleotide of SEQ ID NO:404.

The 4D13 peptide was inserted into the p2JM103 based vector constructed for the expression of BBIt-AV-A13I-L29P-A40K-F50T-V52A (SEQ ID NO:404) with an oligonucleotide cassette having BamHI and SacI restriction sites on the ends using the oligonucleotides:

(SEQ ID NO:392) 5′-GATCCAGATGACGAACCGAGCAAACCTTGCTGTGATCCAGACCCTGA CGATGAGAGCT and (SEQ ID NO:393) 5′-CTCATCGTCAGGGTCTGGATCACAGCAAGGTTTGCTCGGTTCGTCAT CTG.

Amino acid sequence of BBIt-AV-4D13 insert-A13I-L29P-A40K-F50T-V52A:

(SEQ ID NO:413) DPDDEPSKPCCDPDPDDESSKPCCDQCICTKSNPPQCRCSDMRPNSCHSA CKSCKCYNLYGWTCTCADITDFCYEPCKPSE

The other variants were made by cloning synthetic genes into the BamHI and HindIII sites of p2JM103-Ink2. The polynucleotide sequences of each of the synthetic genes and resulting modified amino acid sequences of the modified variant BBIt-AVs are given in Table 4.

TABLE 4 BBIt-AVs comprising substitutions/modifications based on the BBIt-AV-A13I-L29P-A40K-F50T-V52A variant (SEQ ID NO:601) Amino Acid Amino Acid Substitutions Synthetic Gene Sequence of the in BBIt-AV Sequence Encoding the BBIt-AV Variant BBIt-AV Variant BBIt-AV-D1C- GGATCCATGCGATGAGAGCTCTAAACCTTGT DPCDESSKPCCDQ A13I-L29P- TGCGATCAATGCATTTGTACTAAATCAAACCC CICTKSNPPQCRCS A40K-F50T- TCCACAATGTCGTTGTTCTGATATGCGTCCTA DMRPNSCHSACKS V52A ATTCATGTCATTCTGCATGCAAATCATGTAAA CKCYNLYGWTCTCA TGCTATAACCTTTACGGTTGGACATGTACATG DITDFCYEPCKPSE CGCAGATATCACTGACTTCTGCTATGAACCAT (SEQ ID NO:611) GTAAACCTTCTGAATAAAAGCTT (SEQ ID NO:414) BBIt-AV-S4V- GGATCCAGACGATGAGGTTTCTAAACCTTGT DPDDEVSKPCCDQ A13I-L29P- TGCGATCAATGCATTTGTACTAAATCAAACCC CICTKSNPPQCRCS A40K-F50T- TCCACAATGTCGTTGTTCTGATATGCGTCCTA DMRPNSCHSACKS V52A ATTCATGTCATTCTGCATGCAAATCATGTAAA CKCYNLYGWTCTCA TGCTATAACCTTTACGGTTGGACATGTACATG DITDFCYEPCKPSE CGCAGATATCACTGACTTCTGCTATGAACCAT (SEQ ID NO:612) GTAAACCTTCTGAATAAAAGCTT (SEQ ID NO:415) BBIt-AV-S5P- GGATCCAGACGATGAGAGCCCTAAACCTTGT DPDDESPKPCCDQ A13I-L29P- TGCGATCAATGCATTTGTACTAAATCAAACCC CICTKSNPPQCRCS A40K-F50T- TCCACAATGTCGTTGTTCTGATATGCGTCCTA DMRPNSCHSACKS V52A ATAGCTGTCATTCTGCATGCAAATCATGTAAA CKCYNLYGWTCTCA TGCTATAACCTTTACGGTTGGACATGTACATG DITDFCYEPCKPSE CGCAGATATCACTGACTTCTGCTATGAACCAT (SEQ ID NO:613) GTAAACCTTCTGAATAAAAGCTT (SEQ ID NO:416) BBIt-AV- GGATCCAGACGATGAGAGCTCTAAACCTTGT DPDDESSKPCCDG Q11G-A13I- TGCGATGGCTGCATTTGTACTAAATCAAACC CICTKSNPPQCRCS L29P-A40K- CTCCACAATGTCGTTGTTCTGATATGCGTCCT DMRPNSCHSACKS F50T-V52A AATTCATGTCATTCTGCATGCAAATCATGTAA CKCYNLYGWTCTCA ATGCTATAACCTTTACGGTTGGACATGTACAT DITDFCYEPCKPSE GCGCAGATATCACTGACTTCTGCTATGAACC (SEQ ID NO:614) ATGTAAACCTTCTGAATAAAAGCTT (SEQ ID NO:417) BBIt-AV- GGATCCAGACGATGAGAGCTCTAAACCTTGT DPDDESSKPCCDQ A13L-L29P- TGCGATCAATGCCTTTGTACTAAATCAAACCC CLCTKSNPPQCRCS A40K-F50T- TCCACAATGTCGTTGTTCTGATATGCGTCCTA DMRPNSCHSACKS V52A ATTCATGTCATTCTGCATGCAAATCATGTAAA CKCYNLYGWTCTCA TGCTATAACCTTTACGGTTGGACATGTACATG DITDFCYEPCKPSE CGCAGATATCACTGACTTCTGCTATGAACCAT (SEQ ID NO:615) GTAAACCTTCTGAATAAAAGCTT (SEQ ID NO:418) BBIt-AV-A13I- GGATCCAGACGATGAGAGCTCTAAACCTTGT DPDDESSKPCCDQ S25R-L29P- TGCGATCAATGCATTTGTACTAAATCAAACCC CICTKSNPPQCRCR A40K-F50T- TCCACAATGTCGTTGTAGAGATATGCGTCCT DMRPNSCHSACKS V52A AATAGCTGTCATTCTGCATGCAAATCATGTAA CKCYNLYGWTCTCA ATGCTATAACCTTTACGGTTGGACATGTACAT DITDFCYEPCKPSE GCGCAGATATCACTGACTTCTGCTATGAACC (SEQ ID NO:616) ATGTAAACCTTCTGAATAAAAGCTT (SEQ ID NO:419) BBIt-AV-A13I- GGATCCAGACGATGAGAGCTCTAAACCTTGT DPDDESSKPCCDQ S25R-L29P- TGCGATCAATGCATTTGTACTAAATCAAACCC CICTKSNPPQCRCR S31A-A40K- TCCACAATGTCGTTGTAGAGATATGCGTCCT DMRPNACHSACKS F50T-V52A AATGCTTGTCATTCTGCATGCAAATCATGTAA CKCYNLYGWTCTCA ATGCTATAACCTTTACGGTTGGACATGTACAT DITDFCYEPCKPSE GCGCAGATATCACTGACTTCTGCTATGAACC (SEQ ID NO:617) ATGTAAACCTTCTGAATAAAAGCTT (SEQ ID NO:420) BBIt-AV-A13I- GGATCCAGACGATGAGAGCTCTAAACCTTGT DPDDESSKPCCDQ S25R-L29P- TGCGATCAATGCATTTGTACTAAATCAAACCC CICTKSNPPQCRCR S31R-A40K- TCCACAATGTCGTTGTAGAGATATGCGTCCT DMRPNRCHSACKS F50T-V52A AATCGCTGTCATTCTGCATGCAAATCATGTAA CKCYNLYGWTCTCA ATGCTATAACCTTTACGGTTGGACATGTACAT DITDFCYEPCKPSE GCGCAGATATCACTGACTTCTGCTATGAACC (SEQ ID NO:618) ATGTAAACCTTCTGAATAAAAGCTT (SEQ ID NO:421) BBIt-AV-A13I- GGATCCAGACGATGAGAGCTCTAAACCTTGT DPDDESSKPCCDQ M27R-L29P- TGCGATCAATGCATTTGTACTAAATCAAACCC CICTKSNPPQCRCS A40K-F50T- TCCACAATGTCGTTGTTCTGATAGACGTCCTA DRRPNSCHSACKSC V52A ATTCATGTCATTCTGCATGCAAATCATGTAAA KCYNLYGWTCTCAD TGCTATAACCTTTACGGTTGGACATGTACATG ITDFCYEPCKPSE CGCAGATATCACTGACTTCTGCTATGAACCAT (SEQ ID NO:619) GTAAACCTTCTGAATAAAAGCTT (SEQ ID NO:422) BBIt-AV-A13I- GGATCCAGACGATGAGAGCTCTAAACCTTGT DPDDESSKPCCDQ L29K-A40K- TGCGATCAATGCATTTGTACTAAATCAAACCC CICTKSNPPQCRCS F50T-V52A TCCACAATGTCGTTGTTCTGATATGCGTAAAA DMRKNSCHSACKS ATTCATGTCATTCTGCATGCAAATCATGTAAA CKCYNLYGWTCTCA TGCTATAACCTTTACGGTTGGACATGTACATG DITDFCYEPCKPSE CGCAGATATCACTGACTTCTGCTATGAACCAT (SEQ ID NO:620) GTAAACCTTCTGAATAAAAGCTT (SEQ ID NO:423) BBIt-AV-A13I- GGATCCAGACGATGAGAGCTCTAAACCTTGT DPDDESSKPCCDQ L29P-S31A- TGCGATCAATGCATTTGTACTAAATCAAACCC CICTKSNPPQCRCS A40K-F50T- TCCACAATGTCGTTGTTCTGATATGCGTCCTA DMRPNACHSACKS V52A ATGCTTGTCATTCTGCATGCAAATCATGTAAA CKCYNLYGWTCTCA TGCTATAACCTTTACGGTTGGACATGTACATG DITDFCYEPCKPSE CGCAGATATCACTGACTTCTGCTATGAACCAT (SEQ ID NO:621) GTAAACCTTCTGAATAAAAGCTT (SEQ ID NO:424) BBIt-AV-A13I- GGATCCAGACGATGAGAGCTCTAAACCTTGT DPDDESSKPCCDQ L29P-S31R- TGCGATCAATGCATTTGTACTAAATCAAACCC CICTKSNPPQCRCS A40K-F50T- TCCACAATGTCGTTGTTCTGATATGCGTCCTA DMRPNRCHSACKS V52A ATAGATGTCATTCTGCATGCAAATCATGTAAA CKCYNLYGWTCTCA TGCTATAACCTTTACGGTTGGACATGTACATG DITDFCYEPCKPSE CGCAGATATCACTGACTTCTGCTATGAACCAT (SEQ ID NO:622) GTAAACCTTCTGAATAAAAGCTT (SEQ ID NO:425) BBIt-AV-A13I- GGATCCAGACGATGAGAGCTCTAAACCTTGT DPDDESSKPCCDQ L29P-S38N- TGCGATCAATGCATTTGTACTAAATCAAACCC CICTKSNPPQCRCS A40K-F50T- TCCACAATGTCGTTGTTCTGATATGCGTCCTA DMRPNSCHSACKN V52A ATAGCTGTCATTCTGCATGCAAAAACTGTAAA CKCYNLYGWTCTCA TGCTATAACCTTTACGGTTGGACATGTACATG DITDFCYEPCKPSE CGCAGATATCACTGACTTCTGCTATGAACCAT (SEQ ID NO:623) GTAAACCTTCTGAATAAAAGCTT (SEQ ID NO:426) BBIt-AV-A13I- GGATCCAGACGATGAGAGCTCTAAACCTTGT DPDDESSKPCCDQ L29P-A40K- TGCGATCAATGCATTTGTACTAAATCAAACCC CICTKSNPPQCRCS F50K-V52A TCCACAATGTCGTTGTTCTGATATGCGTCCTA DMRPNSCHSACKS ATTCATGTCATTCTGCATGCAAATCATGTAAA CKCYNLYGWTCKC TGCTATAACCTTTACGGTTGGACATGTAAATG ADITDFCYEPCKPSE CGCAGATATCACTGACTTCTGCTATGAACCAT (SEQ ID NO:624) GTAAACCTTCTGAATAAAAGCTT (SEQ ID NO:427) BBIt-AV-A13I- GGATCCAGACGATGAGAGCTCTAAACCTTGT DPDDESSKPCCDQ L29P-A40K- TGCGATCAATGCATTTGTACTAAATCAAACCC CICTKSNPPQCRCS F50T-V52T TCCACAATGTCGTTGTTCTGATATGCGTCCTA DMRPNSCHSACKS ATTCATGTCATTCTGCATGCAAATCATGTAAA CKCYNLYGWTCTCT TGCTATAACCTTTACGGTTGGACATGTACATG DITDFCYEPCKPSE CACAGATATCACTGACTTCTGCTATGAACCAT (SEQ ID NO:625) GTAAACCTTCTGAATAAAAGCTT (SEQ ID NO:428) BBIt-AV-A13I- GGATCCAGACGATGAGAGCTCTAAACCTTGT DPDDESSKPCCDQ L29P-A40K- TGCGATCAATGCATTTGTACTAAATCAAACCC CICTKSNPPQCRCS F50T-V52A- TCCACAATGTCGTTGTTCTGATATGCGTCCTA DMRPNSCHSACKS S65E ATAGCTGTCATTCTGCATGCAAATCATGTAAA CKCYNLYGWTCTCA TGCTATAACCTTTACGGTTGGACATGTACATG DITDFCYEPCKPEE CGCAGATATCACTGACTTCTGCTATGAACCAT (SEQ ID NO:626) GTAAACCTGAAGAATAAAAGCTT (SEQ ID NO:429)

The BBI:BCE ratio of the modified variant BBIt-AVs was determined as described above, and the yield for each of the modified variant BBIt-AVs was calculated. The modified BBIt-AVs BBIt-AV-A13I-S25R-L29P-S31R-A40K-F50T-V52A (SEQ ID NO:618), BBIt-AV-A13I-S25R-L29P-S31A-A40K-F50T-V52A (SEQ ID NO:617), BBIt-AV-A13I-S25R-L29P-A40K-F50T-V52A (SEQ ID NO:616), BBIt-AV-A13I-L29P-S31R-A40K-F50T-V52A (SEQ ID NO:622) had significantly better yields after acid/heat treatment than quintuple BBIt-AV-A13I-L29P-A40K-F50T-V52A (SEQ ID NO:601), while BBIt-AV-A13I-L29P-A40K-F50K-V52A (SEQ ID NO:624), BBIt-AV-A13L-L29P-A40K-F50T-V52A (SEQ ID NO:615) and BBIt-AV-4D13 insert-A13I-L29P-A40K-F50T-V52A (SEQ ID NO:413) had slightly better yields than BBIt-AV-A13I-L29P-A40K-F50T-V52A (SEQ ID NO:601). The modified BBIt-AV-A13I-L29P-A40K-F50T-V52A-S65E (SEQ ID NO:626) and BBIt-AV-A13I-L29P-S38N-A40K-F50T-V52A (SEQ ID NO:623) had similar yields as BBIt-AV-A13I-L29P-A40K-F50T-V52 (SEQ ID NO:624), BBIt-AV-A13I-M27R-L29P-A40K-F50T-V52A (SEQ ID NO:619), BBIt-AV-A31I-L29P-S31A-A40K-F50T-V52A (SEQ ID NO:621) and BBIt-AV-A13I-L29P-A40K-F50T-V52T (SEQ ID NO:615) somewhat less, while BBIt-AV-S4V-A31I-L29P-A40K-F50T-V52A (SEQ ID NO:612), BBIt-AV-Q11G-A13I-L29P-A40K-F50T-V52A (SEQ ID NO:614), BBIt-AV-A13I-L29K-A40K-F50T-V52A (SEQ ID NO:620) and BBIt-AV-S5P-A13I-L29P-A40K-F50T-V52A (SEQ ID NO:611) all had significantly lower yields than BBIt-AV-A13I-L29P-A40K-F50T-V52A (SEQ ID NO:601), after acid-heat treatment. The yield of BBIt-AV-D1C-A13I-L29P-A40K-F50T-V52A (SEQ ID NO:611) in the absence of activation was also much lower than BBIt-AV-A13I-L29P-A40K-F50T-V52A (SEQ ID NO:601). However, the yield after acid/heat treatment of BBIt-AV-D1C-A13I-L29P-A40K-F50T-V52A (SEQ ID NO:611) when not activated was about two fold higher than BBIt-AV-A13I-L29P-A40K-F50T-V52A (SEQ ID NO:601) when activated but similar to BBIt-AV-A13I-L29P-A40K-F50T-V52A (SEQ ID NO:601) when it was not activated. Thus, no substitutions were found that could produce high yields without activation.

Generation of Modified BBI-AVs Containing Eight Amino Acid Substitutions: Octuple Variants

Following the testing of the quintuple-based modified BBI-AVs, additional modified variant BBIs (octuple variants) were generated to contain eight amino acid substitutions. The activity and purification yield of the octuple variants were determined as described above. Construction of the octuple modified BBIt-AVs was achieved as described above using the synthetic genes shown in Table 5.

TABLE 5 Octuple Modified Variant BBIt-AVs Amino Acid Synthetic Gene Sequence Amino Acid Sequence Substitutions Encoding the Octuple Variant of the BBIt-AV Variant BBIt-AV-A131- GGATCCAGACGATGAGAGCTCTAAACCTT DPDDESSKPCCDQCICTKSNP S25R-M27A- GTTGCGATCAATGCATCTGTACAAAATCA PQCRCRDARPNACHSACKSC L29P-S31A- AACCCTCCACAATGTCGTTGTAGAGATGC HCYNLYGWTCKCTDITDFCYE A40H-F50K- TCGTCCTAATGCATGTCATTCTGCATGCA PCKPSE V52T (KT8) AATCATGTCACTGCTATAACCTTTACGGTT (SEQ ID NO:627) GGACATGTAAATGCACAGACATCACTGAC TTCTGCTATGAACCATGTAAACCTTCTGAA TAAAAGCTT (SEQ ID NO:486) BBIt-AV-A131- GGATCCAGACGATGAGAGCTCTAAACCTT DPDDESSKPCCDQCICTKSNP S25K-M27A- GTTGCGATCAATGCATCTGTACAAAATCA PQCRCKDARRNECHSACKSC L29R-S31E- AACCCTCCACAATGTCGTTGTAAAGATGC KCYNLYGWTCQCQDITDFCYE A40K-F50Q- TCGTAGAAATGAATGTCATTCTGCATGCA PCKPSE V52Q (QQ8) AATCATGTAAATGCTATAACCTTTACGGTT (SEQ ID NO:628) GGACATGTCAATGCCAAGACATCACTGAC TTCTGCTATGAACCATGTAAACCTTCTGAA TAAAAGCTT (SEQ ID NO:487) BBIt-AV-A131- GGATCCAGACGATGAGAGCTCTAAACCTT DPDDESSKPCCDQCICTKSNP S25K-M27R- GTTGCGATCAATGCATCTGTACAAAATCA PQCRCKDRRENACHSACKSC L29E-S31A- AACCCTCCACAATGTCGTTGTAAAGATAG HCYNLYGWTCRCKDITDFCYE A40H-F50R- ACGTGAAAATGCTTGTCATTCTGCATGCA PCKPSE V52K (RK8) AATCATGTCACTGCTATAACCTTTACGGTT (SEQ ID NO:629) GGACATGTAGATGCAAAGACATCACTGAC TTCTGCTATGAACCATGTAAACCTTCTGAA TAAAAGCTT (SEQ ID NO:488) BBIt-AV-A131- GGATCCAGACGATGAGAGCTCTAAACCTT DPDDESSKPCCDQCICTKSNP S25K-M27A- GTTGCGATCAATGCATCTGTACAAAATCA PQCRCKDARRNACHSACKSC L29R-S31A- AACCCTCCACAATGTCGTTGTAAAGATGC HCYNLYGWTCRCLDITDFCYE A40H-F50R- TCGTAGAAATGCTTGTCATTCTGCATGCA PCKPSE V52L (RL8) AATCATGTCACTGCTATAACCTTTACGGTT (SEQ ID NO:630) GGACATGTAGATGCTTAGACATCACTGAC TTCTGCTATGAACCATGTAAACCTTCTGAA TAAAAGCTT (SEQ ID NO:489) BBIt-AV-A131- GGATCCAGACGATGAGAGCTCTAAACCTT DPDDESSKPCCDQCICTKSNP S25K-M27Q- GTTGCGATCAATGCATCTGTACAAAATCA PQCRCKDQRPNECHSACKSC L29P-F50R- AACCCTCCACAATGTCGTTGTAAAGATCA HCYNLYGWTCRCQDITDFCYE A40H-F50R- ACGTCCTAATGAATGTCATTCTGCATGCA PCKPSE V52Q (RQ8) AATCATGTCACTGCTATAACCTTTACGGTT (SEQ ID NO:631) GGACATGTAGATGCCAAGACATCACTGAC TTCTGCTATGAACCATGTAAACCTTCTGAA TAAAAGCTT (SEQ ID NO:490)

The best octuple variants selected in the shake flask screen were tested in the purification process essentially as described above, and including the following changes: 1) after growth, the culture was two fold diluted in glycine buffer (125 mM final concentration and pH adjusted to 9.0) instead of CHES buffer; 2) samples were activated with 2 mM 2-mercaptoethanol only (no sodium sulfite was added; 3) the acid precipitated fusion protein was resuspended in 100 ml of 125 mM glycine (rather than 350 ml of 40 mM glycine); and 4) the pellet collected after the acid/heat treatment was washed with 10 ml water, filtered, and this “washed pellet” filtrate was combined with the original filtrate. The final filtrates were concentrated and trimmed with Glu-BL as described above

The results show that octuple variant BBIt-AV molecules QQ8 (SEQ ID NO:628) and KT8 (SEQ ID NO:627) resulted in purification yields that were greater than that of the quintuple modified variant BBIt-AV-A13I-L29P-A40K-F50T-V52A variant (SEQ ID NO:601). The modified octuple variants BBIt-AV-A13I-S25K-M27A-L29R-S31A-A40H-F50R-V52L (RL8; SEQ ID NO:630), BBIt-AV-A13I-S25K-M27A-L29R-S31E-A40K-F50Q-V52Q (QQ8; SEQ ID NO:628) and BBIt-AV-A13I-S25R-M27A-L29P-S31A-A40H-F50K-V52T (KT8; SEQ ID NO:627) resulted in yields that were better than the wild-type sBBI molecule/protein (FIG. 13).

Example 13 Improved Production of BBI Scaffolds Carrying FGF5 or TGFβ1 Binding Peptides

In this example, the same amino acid substitutions that resulted in modified variant BBIt-AVs having improved trypsin inhibitory activity and/or purification yields (see Examples 7-13) were tested for improving the trypsin inhibitory activity and/or the purification yield of variant BBI scaffolds in which the chymotrypsin loop was replaced with either an FGF-5 or a TGFβ1 binding peptide.

Generation of Variant and Modified Variant BBI-FGF-5 Proteins:

Modified variant BBIt-FGF-5 protease inhibitors comprising the combination of substitutions A13I-L29P-F50T-V52A (all with alanine at position 40) and either the FGF-5 binding peptide MM007: RTQPYPL (SEQ ID NO:670), or FGF-5 binding peptide FGFps2: TWIDSTP (SEQ ID NO:671) in place of the chymotrypsin loop, were constructed by ligating a synthetic gene into the BamHI and HindIII sites of the vector p2JMagk103-Ink2-BBIt-AV. The amino acid sequences and DNA sequences of the synthetic genes encoding the resulting modified variant MM007-Q-BBIt-FGF-5 (SEQ ID NO:432; SEQ ID NO:433) and the modified variant FGFps2-Q-BBIt-FGF-5, respectively (SEQ ID NO:434 SEQ ID NO:435) are as follows.

MM007-Q-BBIt-FGF-5 synthetic gene (SEQ ID NO:433) GGATCCAGACGATGAGAGCTCTAAACCTTGTTGCGATCAATGCATTTGTA CTAAATCAAATCCTCCACAATGTCGTTGTTCTGATATGCGTCCTAATAGC TGTCATTCTGCATGCAAATCATGTGCTTGCCGTACTCAACCATACCCTCT TTGTACATGCGCAGACATCACTGACTTCTGCTATGAACCATGTAAACCAT CTGAATAAAAGCTT MM007-Q-BBIt-FGF-5 protein (SEQ ID NO:432) DPDDESSKPCCDQCICTKSNPPQCRCSDMRPNSCHSACKSCACRTQPYPL CTCADITDFCYEPCKPSE FGFps2-Q-BBIt-FGF-5 (SEQ ID NO:435) GGATCCAGACGATGAGAGCTCTAAACCTTGTTGCGATCAATGCATTTGTA CTAAATCAAATCCTCCACAATGTCGTTGTTCTGATATGCGTCCTAATAGC TGTCATTCTGCATGCAAATCATGTGCTTGCACTTGGATTGATTCAACACC ATGTACATGCGCAGACATCACTGACTTCTGCTATGAACCATGTAAACCAT CTGAATAAAAGCTT (SEQ ID NO:434) DPDDESSKPCCDQCICTKSNPPQCRCSDMRPNSCHSACKSCACTWIDSTP CTCADITDFCYEPCKPSE

Generation of Variant and Modified Variant BBI-TGFβ Proteins:

The chymotrypsin inhibitory loop of the BBIt-AV of SEQ ID NO:187 was replaced with the TGFβ1 binding peptide PEN3: CLCPENINVLPCN (SEQ ID NO:436), the TGFβ1 binding peptide MM021W: CICKHNVDWLCF (SEQ ID NO:437) or the TGFβ1 binding peptide WTQ: CICWTQHIHNCF (SEQ ID NO:438) to generate variant BBPIs according to the method described in Example 2, as follows.

Oligonucleotide pairs were used to make cassettes to replace the FGFhI binding peptide in the chymotrypsin reactive site loop encoded by the p2JM103-Ink2-2BBI-FGFhI expression vector with the TGFβ1 binding peptides, PEN3, MM021W or WTQ. The p2JM103-Ink2-2BBI-FGFhI was constructed using the primers (SEQ ID NO:91 and SEQ ID NO:92) and QuikChange® method as described in Example 2. The TGFβ binding cassettes were ligated into the SphI and SalI restriction sites in the vector p2JM103Ink2-2BBI-FGFhI to construct the variant BBI molecules PEN3-BBIt-TGFβ₁, MM021W-BBIt-TGFβ and WTQ-BBIt-TGFβ₁, respectively. The DNA sequences of the oligonucleotides used to make the cassettes are shown below.

PEN3 (2^(nd) loop) (SEQ ID NO:439) CAAAAGCTGTCTTTGTCCTGAAAATATTAACGTTCTTCCTTGTAACTGCG And (SEQ ID NO:440) TCGACGCAGTTACAAGGAAGAACGTTAATATTTTCAGGACAAAGACAGCT TTTGCATG MM021W (2^(nd) loop) (SEQ ID NO:129) CAAATCATGCATTTGTAAACACAACGTAGATTGGTTATGTTTTTGCG And (SEQ ID NO:130) TCGACGCAAAAACATAACCAATCTACGTTGTGTTTACAAATGCATGATTT GCATG WTQ (2^(nd) loop) (SEQ ID NO:441) CAAATCATGCATTTGTTGGACACAACATATCCACAACTGTTTTTGCG And (SEQ ID NO:442) TCGACGCAAAAACAGTTGTGGATATGTTGTGTCCAACAAATGCATGATTT GCATG

In addition, modified variant BBPI-TGFβ1 variants comprising the combination of substitutions A13I-L29P-V52A (all also have alanine at position 40 and threonine at position 50) and the TGFβ1 binding peptide PEN3-Q: CPENINVLPC (SEQ ID NO:672), MM021W-Q: CKHNVDWLC (SEQ ID NO:673) or WTQ-Q: CWTQHIHNC (SEQ ID NO:674) in place of the chymotrypsin loop, were constructed by ligating a synthetic gene into the BamHI and HindIII sites of the vector p2JMagk103-Ink2-BBIt-AV. The amino acid sequences and DNA sequences of the synthetic genes encoding the resulting modified variant PEN3-Q-BBIt-TGFβ1 (SEQ ID NO:443; SEQ ID NO:444), the modified variant MM021W-BBI-TGF β1 (SEQ ID NO:445; SEQ ID NO:446), and the modified variant WTQ-BBI-TGFβ1 (SEQ ID NO:447; SEQ ID NO:448) are as follows.

PEN3-Q-BBIt⊕1 (SEQ ID NO:443) DPDDESSKPCCDQCICTKSNPPCQCRCSDMRPNSCHSACKSCACPENINV LPCTCADITDFCYEPCKPSE PEN3-Q-BBIt-TGFβ1 (SEQ ID NO:444) GGATCCAGACGATGAGAGCTCTAAACCTTGTTGCGATCAATGCATTTGTA CTAAATCAAATCCTCCACAATGTCGTTGTTCTGATATGCGTCCTAATAGC TGTCATTCTGCATGCAAATCATGTGCTTGCCCAGAAAACATCAACGTTCT TCCTTGTACATGCGCAGACATCACTGACTTCTGCTATGAACCATGTAAAC CATCTGAATAAAAGCTT MM021W-Q-BBIt-TGFβ1 (SEQ ID NO:445) DPDDESSKPCCDQCICTKSNPPQCRCSDMRPNSCHSACKSCACKHNVDWL CTCADITDFCYEPCKPSE MM021W-Q-BBIt-TGFβ1 (SEQ ID NO:446) GGATCCAGACGATGAGAGCTCTAAACCTTGTTGCGATCAATGCATTTGTA CTAAATCAAATCCTCCACAATGTCGTTGTTCTGATATGCGTCCTAATAGC TGTCATTCTGCATGCAAATCATGTGCTTGCAAACATAACGTTGATTGGCT TTGTACATGCGCAGACATCACTGACTTCTGCTATGAACCATGTAAACCAT CTGAATAAAAGCTT WTQ-Q-BBIt-TGFβ1 (SEQ ID NO:447) DPDDESSKPCCDQCICTKSNPPQCRCSDMRPNSCHSACKSCACKHNVDWL CTCADITDFCYEPCKPSE WTQ-Q-BBIt-TGFβ1 (SEQ ID NO:448) GGATCCAGACGATGAGAGCTCTAAACCTTGTTGCGATCAATGCATTTGTA CTAAATCAAATCCTCCACAATGTCGTTGTTCTGATATGCGTCCTAATAGC TGTCATTCTGCATGCAAATCATGTGCTTGCTGGACACAACATATCCACAA CTGTACATGCGCAGACATCACTGACTTCTGCTATGAACCATGTAAACCAT CTGAATAAAAGCTT

The ten vectors comprising the variant and modified variant BBIs containing an FGF-5 or a TGFβ binding peptide were used to transform B. subtilis BG6006 host cells. The transformants were grown and the engineered BBIs produced by the host cells were tested for trypsin inhibitory activities and production yields as described above.

FIG. 14 shows the trypsin inhibitory activity of the unmodified variant parent MM007-PT-BBIt-FGF-5 (SEQ ID NO:678): (DDESSKPCCDQCACTKSNPPQCRCSDMRLNSCHSACKSCAC RTQPYPL CFCVDITDFCYEPCKPSE; SEQ ID NO:678), the unmodified variant parent FGFps2-PT-BBIt-FGF5 (SEQ ID NO:679): (DDESSKPCCDQCACTKSNPPQCRCSDMRLNSCHSACKSCAC TWIDSTP CFCVDITDFCYEPCKPSE; SEQ ID NO:679), the unmodified variant parent PEN3-PT-BBIt-TGFβ₁ (SEQ ID NO:680): (DDESSKPCCDQCACTKSNPPQCRCSDMRLNSCHSACKSCAC PENINVLP CFCVDITDFCYEPCKPSE; SEQ ID NO:680), the unmodified variant parent WTQ-PT-TGFβ₁ (SEQ ID NO:681): (DDESSKPCCDQCACTKSNPPQCRCSDMRLNSCHSACKSCAC WTQHIHNCF CFCVDITDFCYEPCKPSE; SEQ ID NO:681), and the unmodified variant parent MM0021W-PT-TGFβ₁ (SEQ ID NO:682): (DDESSKPCCDQCACTKSNPPQCRCSDMRLNSCHSACKSCAC KHNVDWL CFCVDITDFCYEPCKPSE; SEQ ID NO:682), (designated PT in FIG. 14) and the corresponding modified variant MM007-Q-BBIt-FGF-5 (SEQ ID NO:432), the modified variant FGFps2-Q-BBIt-FGF5 (SEQ ID NO:434), the modified variant PEN3-Q-BBIt-TGFβ1 (SEQ ID NO:443), the modified variant WTQ-Q-BBIt-TGFβ1 (SEQ ID NO:445), and the modified variant MM0021W-Q-BBIt-TGFβ1 (SEQ ID NO:447) proteins (designated Q in FIG. 14) measured after growth, after activation with 2-mercaptoethanol and after acid/heat treatment. The data show that the combination of the amino acids 13I-29P-40A-50T-52A when present in the BBIt scaffold carrying FGF-5 or TGFβ1 binding peptides improve the trypsin inhibitory activity after growth and activation, and the production yield after acid/heat treatment when compared to the corresponding parent BBI scaffold carrying the binding peptides.

The data also evidences that substitutions that were shown to improve the trypsin inhibitory activity and/or purification yield of a modified variant BBI in which the chymotrypsin loop had been replaced with a VEGF binding peptide are not specific to BBI-AVs as the same substitutions improve the trypsin inhibitory activity and/or the production yield of BBIs in which the chymotrypsin loop was replaced by other binding peptides e.g. FGF-5 binding peptides and TGFβ1 binding peptides.

Therefore, the amino acid substitutions made in the BBI scaffold that increase the production yield and/or the trypsin activity of BBIt-AV are expected to be generally applicable to BBI scaffolds carrying other binding peptides.

Example 14 Performance of Bowman Birk Inhibitor Scaffolds

In this example, the ability to predict the effect of amino acid substitutions on the trypsin activity and/or production yield of a variant Bowman Birk Inhibitor e.g. BBIt-AV (SEQ ID NO:187) was tested by substituting amino acids that are not conserved across Bowman Birk Inhibitor sequences from Dolichos biflorus (BBdb, Acc. No. AAK97765; SEQ ID NO:449) PSESSKPCCDQCACTKSIPPQCRCTDVRLNSCHSACSSCVCTFSIPAQCVCVDMKDFCYEPCK (SEQ ID NO:449), from Glycine max (soybean) protease inhibitor IV or D-II (BBsb3, Acc. No. P01064; SEQ ID NO: 450) DDEYSKPCCDLCMCTRSMPPQCSCEDIRLNSCHSDCKSCMCTRSQPGQCRCLDT NDFCYKPCKSRDD (SEQ ID NO:450), and from Torresea (Amburana) cearensis (BBtc, Acc. No. P83283; SEQ ID NO:451) SSKWEACCDRCACTKSIPPQCHCADIRLNSCHSACESCACTHSIPAQCRCFDITDFCYKPCSG (SEQ ID NO:451), into the BBIt-AV scaffold.

Variant Bowman Birk Inhibitors were generated by replacing the second protease inhibitory loop of the Bowman Birk Inhibitor from Dolichos biflorus (BBdb, Acc. No. AAK97765; SEQ ID NO:449), the second protease inhibitory loop of the Bowman Birk Inhibitor from Glycine max (soybean) protease inhibitor IV or D-II (BBsb3, Acc. No. P01064; SEQ ID NO: 450) and the second protease inhibitory loop of the Bowman Birk Inhibitor from Torresea (Amburana) cearensis (BBtc, Acc. No. P83283; SEQ ID NO:451) with the VEGF binding peptide CK37281 (ACYNLYGWTC; SEQ ID NO:9) to generate the corresponding variant BBdb-AV (SEQ ID NO:452), BBsb3-AV (SEQ ID NO:453), and BBtc-AV (SEQ ID NO:454).

The sequences were aligned, as shown in FIG. 15, and amino acids at positions equivalent to positions 11, 13, 18, 23, 25, 27, 35, 37, 52, 54 and 55 of the BBPI of SEQ ID NO:187 (BBIt-AV) were identified as not being conserved across the four inhibitors, and were chosen to modify the BBIt-AV scaffold.

The synthetic genes encoding BBdb-AV (SEQ ID NO:455) and BBsb3-AV (SEQ ID NO:456) were ligated into the p2JMagk103-Ink2-BBIt-AV as BamHI-HindIII fragments. For cloning the BBtc-AV gene, the gene was first digested with BfrI, the overhang filled in with T4 DNA polymerase and dNTP's, the DNA purified, and then digested with BamHI. This fragment was ligated into the p2JMagk103-Ink2-BBIt-AV vector that had been digested with HindIII, the overhang filled in with T4 DNA polymerase and dNTP's, the DNA purified, and then digested with BamHI. The DNA sequences and the corresponding amino acid sequences of the variant PIs are as follows.

BBdb-AV (SEQ ID NO:455) GGATCCTTCTGAGAGCTCTAAACCATGCTGTGATCAATGCGCTTGTACAA AATCTATCCCTCCACAATGCCGTTGCACTGATGTTCGTCTTAACTCATGT CACTCTGCATGCAGCTCATGCGCTTGTTACAACCTTTACGGTTGGACATG CGTTTGCGTCGACATGAAAGATTTCTGCTACGAACCTTGTAAATAAAAGC TT BBdb-AV (SEQ ID NO:452) DPSESSKPCCDQCACTKSIPPQCRCTDVRLNSCHSACSSCACYNLYGWTC VCVDMKDFCYEPCK BBsb3-AV (SEQ ID NO:456) GGATCCAGATGACGAATACTCTAAACCTTGCTGTGATCTTTGCATGTGTA CACGTTCTATGCCACCTCAATGCTCATGTGAAGACATCCGCCTTAACTCT TGCCACTCAGATTGCAAAAGCTGCGCTTGTTACAACCTTTACGGTTGGAC ATGCCGTTGTTTAGATACTAACGATTTCTGCTACAAACCTTGCAAATCTC GTGATGATTAAAAGCTT BBsb3-AV AA (SEQ ID NO:453) DPDDEYSKPCCDLCMCTRSMPPQCSCEDIRLNSCHSDCKSCACYNLYGWT CRCLDTNDFCYKPCKSRDD BBtc-AV (SEQ ID NO:457) GGATCCTTCTTCAAAATGGGAAGCTTGCTGTGATCGTTGCGCATGCACAA AATCTATCCCTCCACAATGCCACTGCGCTGATATCCGTCTTAACTCATGC CATTCTGCATGCGAAAGCTGCGCTTGTTACAACCTTTACGGTTGGACATG CCGTTGCTTCGATATCACTGATTTCTGTTACAAACCTTGCTCTGGCTAAA AGCTTAAAAGGAGACCGTTAATCTAAAATCATTATTTGAGGCCCGAGCTT AAAGCTTAAG BBtc-AV AA (SEQ ID NO:454) DPSSKWEACCDRCACTKSIPPQCHCADIRLNSCHSACESCACYNLYGWTC RCFDITDFCYKPCSG

The three expression vectors comprising the variant inhibitor sequences were used to transform B. subtilis host cells BG6006.

Typically, substitutions at the N-terminal and C-terminal ends, which are outside of the first disulfide bond (C8-C62), have small effects on the trypsin inhibitory activity and/or purification yield. Thus, only substitutions of amino acids at positions between C8 and C62 were studied. The substitutions targeted in this study are shown in FIG. 15 (bold and underlined).

The individual amino acid substitutions at positions 11, 13, 18, 23, 25, 27, 35, 37, 50, 52, 54, 55, and 60 in BBIt-AV (SEQ ID NO:187) were constructed as described in Examples 8 and 10. These substitutions were analyzed by screening in microtiter plates (with and without reducing agent) as described in Examples 9 and 10. The results are summarized in Table 6 below

TABLE 6 Effect of single amino acid substitutions present in the BBtc-AV, BBdb-AV and BBsb3-AV scaffolds on the trypsin activity of the modified variant BBIt-AV Amino acid present in the alternative scaffold and relative activity of the amino acid substitution Position and amino BBItc-AV BBIdb-AV BBIsb3-AV acid present in the (SEQ ID (SEQ ID (SEQ ID BBIt-AV scaffold NO: 454) NO: 452) NO: 453) Q11 R (+/−) wt* L (+/−)** A13 wt wt M (+)*** N18 I (+++) I (+++) M (+/−) R23 H (−) wt S (−−−) S25 A (+) T (+/−) E (−−−) M27 I (+/−) V (+/−) I (+/−) A35 wt wt D (+/−) K37 E (+/−) S (+/−) wt F50 R (++) V (+) R (++) V52 F (+/−) wt L (+) I54 wt M (−) T (−)**** T55 wt K (−) N (+/−) E60 K (+/−) wt K (+/−) *wt indicates that the amino acid at this position is the same as in the BBIt-AV scaffold (SEQ ID NO: 187) **(+/−) indicates that the relative BBI:BCE activity ratio in the presence of reducing agent is not significantly different than that of BBIt-AV. ***(+) indicates that the the relative BBI:BCE activity ratio in the presence of reducing agent is greater than that of BBIt-AV; the greater the number of “+”, the greater the difference. ****(−)indicates that the the relative BBI:BCE activity ratio in the presence of reducing agent is less than that of BBIt-AV; the greater the number of “−”, the greater the difference.

Assuming the effects of the amino acid substitutions are additive, the data provided in Table 6 predicts that the variant BBItc-AV would perform significantly better than BBIt-AV, while BBIsb3-AV, should perform significantly worse than BBIt-AV. In addition, one would predict that BBIdb-AV would perform somewhat better than BBIt-AV in terms of activation with reducing agent.

To test these predictions, the trypsin activity and production yield of BBIdb-AV, BBIsb3-AV and BBItc-AV were tested using the Shake Flask Screen described in Example 11 and their activity compared to that of the unmodified variant BBIt-AV.

The data shown in FIG. 16 show that, as predicted, BBItc-AV had the highest trypsin inhibitory activity after activation, and BBIdb-AV inhibitory activity was also greater than that of the BBIt-AV inhibitor but less than that of the BBItc-AV inhibitor. In addition, and as predicted, BBIsb3-AV had very low trypsin inhibitory activity and did not activate with reducing agent (inhibitory activity was too small to be shown in FIG. 16). Furthermore, not only did BBItc-AV and BBIdb-AV have higher inhibitory activities after activation than BBIt-AV, they also had higher inhibitory activities after acid/heat treatment indicating improved production yields.

Therefore, the results indicate that activity data obtained when testing single amino acid substitutions in BBIt-AV can be used to predict the performance of other Bowman Birk Inhibitors when used as scaffolds with binding peptides replacing the second protease inhibitory loop

Example 15 Binding of Modified Variant BBIs to Target Proteins

In this example, the capacity of variant peptides to retain their ability to bind the corresponding target proteins was tested when the variant peptides were grafted into a modified Bowman Birk Inhibitor scaffold to replace the chymotrypsin loop.

The construction of the expression vector for BBI is described in Example 1 and the constructions of vectors for variant BBIs containing the VEGF-binding peptide CK37281 (SEQ ID NO:9), the FGF-5-binding peptide MM007 (SEQ ID NO:430) or the FGF-5-binding peptide FGF5ps2 (SEQ ID NO:431) in place of the chymotrypsin inhibitory sequence are described in Example 13 above. The method for the construction of expression vectors encoding for the modified variant BBIt-AVs having various combinations of amino acid substitution is described in Example 12 and the method for constructing the FGF5 and TGFβ-binding BBIs is described in Example 13.

VEGF-binding peptides named VegK (KYYLYWW; SEQ ID NO:458), VegT (TLWKSYW; SEQ ID NO:459) and VegKD (KYDLYWW; SEQ ID NO: 460) were introduced into the chymotrypsin inhibitory loop by ligating oligonucleotide cassettes into the SphI and SalI sites of p2JM103-Ink2-2BBIck81. The sequences of the oligonucleotides used to generate the modified variant VEGK and VEGT BBPIs are shown below.

VegK: (SEQ ID NO:461) CAAATCTTGCGCATGTAAATATTACCTTTACTGGTGGTGTTTTTGCG And (SEQ ID NO:462) TCGACGCAAAAACACCACCAGTAAAGGTAATATTTACATGCGCAAGATTT GCATG BBIT-VEGK (SEQ ID NO:640) DPDDESSKPCCDQCICTKSNPPQCRCRDARPNACHSACKSCACKYYLYWW CKCTDITFDCYEPCKPSE VegT: (SEQ ID NO:463) CAAATCTTGCGCGTGCACACTTTGGAAATCTTACTGGTGTTTTTGCG And (SEQ ID NO:464) TCGACGCAAAAACACCAGTAAGATTTCCAAAGTGTGCACGCGCAAGATTT GCATG BBIt-VEGT (SEQ ID NO:641) DPDDESSKPCCDQCICTKSNPPQCRCRDARPNACHSACKSCACTLWKSYW CKCTDITDFCYEPCKPSE VegKD: (SEQ ID NO:465) CAAATCTTGCATCTGTAAATATGATCTTTACTGGTGGTGTTTTTGCG And (SEQ ID NO:466) TCGACGCAAAAACACCACCAGTAAAGATCATATTTACAGATGCAAGATTT GCATG

The VEGF-binding peptide named VegKD was introduced into the variant BBI-A13I-S25K-L29P-V52K by ligating a synthetic gene (VegKD-Q) into the BamHI and HindIII sites of p2JMagk103-Ink2-2BBIck81. The sequence of the synthetic gene is shown and encoded BBPI are shown below.

VegKD-Q: (SEQ ID NO:467) GGATCCAGACGATGAGAGCTCTAAACCTTGTTGCGATCAATGCATCTGTA CAAAATCAAACCCTCCACAATGTCGTTGTAAAGATATGCGTCCTAATAGC TGTCATTCTGCATGCAAATCATGTATCTGCAAATATGACCTTTACTGGTG GTGTTTCTGCAAAGACATCACTGACTTCTGCTATGAACCATGTAAACCTT CTGAATAAAAGCTT (SEQ ID NO:642) DPDDESSKPCCDQCICTKSNPPQCRCRDARPNACHSACKSCACKYDLYWW CKCTDITDFCYEPCKPSE

The VEGF-binding peptides V1 (SKHSQIT; SEQ ID NO:468), V2 (KTNPSGS; SEQ ID NO:469), V3 (RPTGHSL; SEQ ID NO:470), V4 (KHSAKAE; SEQ ID NO:471), V5 (KPSSASS; SEQ ID NO:472) and V6 (PVTKRVH; SEQ ID NO:473), and the TNFα-binding peptides T1 (RYWQDIP; SEQ ID NO:474), T2 (APEPILA; SEQ ID NO:475) and T3 (DMIMVSI; SEQ ID NO:476), were introduced into the chymotrypsin inhibitory loop of a modified BBIt containing amino acid substitutions A13I-S25R-M27A-L29P-S31A-140A-F50K-V52T by ligating synthetic genes into the BamHI and HindIII sites of p2JMagk103-Ink2-2BBIck81. The DNA sequences of the synthetic genes encoding the resulting modified BBIs are shown below.

BBIt-AV-V1: (SEQ ID NO:477) GGATCCAGACGATGAGAGCTCTAAACCTTGTTGCGATCAATGCATCTGTA CAAAATCAAACCCTCCACAATGTCGTTGTAGAGATGCTCGTCCTAATGCA TGTCATTCTGCATGCAAATCATGTGCTTGCAGCAAACACTCTCAAATTAC TTGTAAATGCACAGACATCACTGACTTCTGCTATGAACCATGTAAACCTT CTGAATAAAAGCTT (SEQ ID NO:491) DPDDESSKPCCDQCICTKSNPPQCRCRDARPNACHSACKSCACSKHSQIT CKCTDITDFCYEPCKPSE BBIt-AV-V2: (SEQ ID NO:478) GGATCCAGACGATGAGAGCTCTAAACCTTGTTGCGATCAATGCATCTGTA CAAAATCAAACCCTCCACAATGTCGTTGTAGAGATGCTCGTCCTAATGCA TGTCATTCTGCATGCAAATCATGTGCTTGCAAAACAAACCCAAGCGGTTC TTGTAAATGCACAGACATCACTGACTTCTGCTATGAACCATGTAAACCTT CTGAATAAAAGCTT (SEQ ID NO:632) DPDDESSKPCCDQCICTKSNPPQCRCRDARPNACHSACKSCACKTNPSGS CKCTDITDFCYEPCKPSE BBIt-AV-V3: (SEQ ID NO:479) GGATCCAGACGATGAGAGCTCTAAACCTTGTTGCGATCAATGCATCTGTA CAAAATCAAACCCTCCACAATGTCGTTGTAGAGATGCTCGTCCTAATGCA TGTCATTCTGCATGCAAATCATGTGCTTGCAGACCAACTGGTCACAGCCT TTGTAAATGCACAGACATCACTGACTTCTGCTATGAACCATGTAAACCTT CTGAATAAAAGCTT (SEQ ID NO:633) DPDDESSKPCCDQCICTKSNPPQCRCRDARPNACHSACKSCACRPTGHSL CKCTDITDFCYEPCKPSE BBIt-AV-V4: (SEQ ID NO:480) GGATCCAGACGATGAGAGCTCTAAACCTTGTTGCGATCAATGCATCTGTA CAAAATCAAACCCTCCACAATGTCGTTGTAGAGATGCTCGTCCTAATGCA TGTCATTCTGCATGCAAATCATGTGCTTGCAAACACAGCGCTAAAGCAGA ATGTAAATGCACAGACATCACTGACTTCTGCTATGAACCATGTAAACCTT CTGAATAAAAGCTT (SEQ ID NO:634) DPDDESSKPCCDQCICTKSNPPQCRCRCARPNACHSACKSCACKHSAKAE CKCTDITDFCYEPCKPSE BBIt-AV-V5: (SEQ ID NO:481) GGATCCAGACGATGAGAGCTCTAAACCTTGTTGCGATCAATGCATCTGTA CAAAATCAAACCCTCCACAATGTCGTTGTAGAGATGCTCGTCCTAATGCA TGTCATTCTGCATGCAAATCATGTGCTTGCAAACCAAGCTCTGCTTCATC TTGTAAATGCACAGACATCACTGACTTCTGCTATGAACCATGTAAACCTT CTGAATAAAAGCTT (SEQ ID NO:635) DPDDESSKPCCDQCICTKSNPPQCRCRDARPNACHSACKSCACKPSSASS CKCTDITDFCYEPCKPSE BBIt-AV-V6: (SEQ ID NO:482) GGATCCAGACGATGAGAGCTCTAAACCTTGTTGCGATCAATGCATCTGTA CAAAATCAAACCCTCCACAATGTCGTTGTAGAGATGCTCGTCCTAATGCA TGTCATTCTGCATGCAAATCATGTGCTTGCCCAGTTACTAAAAGAGTACA CTGTAAATGCACAGACATCACTGACTTCTGCTATGAACCATGTAAACCTT CTGAATAAAAGCTT (SEQ ID NO:636) DPDDESSKPCCDQCICTKSNPPQCRCRDARPNACHSACKSCACPVTKRVH CKCTDITDFCYEPCKPSE BBIt-TNF-T1: (SEQ ID NO:483) GGATCCAGACGATGAGAGCTCTAAACCTTGTTGCGATCAATGCATCTGTA CAAAATCAAACCCTCCACAATGTCGTTGTAGAGATGCTCGTCCTAATGCA TGTCATTCTGCATGCAAATCATGTGCTTGCAGATACTGGCAAGATATTCC ATGTAAATGCACAGACATCACTGACTTCTGCTATGAACCATGTAAACCTT CTGAATAAAAGCTT (SEQ ID NO:637) DPDDESSKPCCDQCICTKSNPPQCRCRDARPNACHSACKSCACRYWQDIP CKCTDITDFCYEPCKPSE BBIt-TNF-T2: (SEQ ID NO:484) GGATCCAGACGATGAGAGCTCTAAACCTTGTTGCGATCAATGCATCTGTA CAAAATCAAACCCTCCACAATGTCGTTGTAGAGATGCTCGTCCTAATGCA TGTCATTCTGCATGCAAATCATGTGCTTGCGCACCAGAACCTATTCTTGC TTGTAAATGCACAGACATCACTGACTTCTGCTATGAACCATGTAAACCTT CTGAATAAAAGCTT (SEQ ID NO:638) DPDDESSKPCCDQCICTKSNPPQCRCRDARPNACHSACKSCACAPEPILA CKCTDITDFCYEPCKPSE BBIt-TNF-T3: (SEQ ID NO:485) GGATCCAGACGATGACαAGCTCTAAACCTTGTTGCGATCAATGCATCTGT ACAAAATCAAACCCTCCACAATGTCGTTGTAGAGATGCTCGTCCTAATGC ATGTCATTCTGCATGCAAATCATGTGCTTGCGATATGATTATGGTTAGCA TCTGTAAATGCACAGACATCACTGACTTCTGCTATGAACCATGTAAACCT TCTGAATAAAAGCTT (SEQ ID NO:639) DPDDESSKPCCDQCICTKSNPPQCRCRDARPNACHSACKSCACDMIMVSI CKCTDITDFCYEPCKPSE

The resulting vectors were used to transform B. subtilis BG6006 host cells. Cultures were grown in 500 ml of MBD medium and the BBI species were purified essentially as described above and by Vogtentanz et al., 2007 (Protein Expr Purif 55:40-52 [2007]). BioVeris binding assays were performed essentially as outlined in Example 5 and by the manufacturer. Binding of the modified variant BBIs to their respective target proteins was measured using a BioVeris® binding assay, which is a competition assay that determines the binding of the modified variant BBIs to its target protein (e.g. VEGF, FGF5, TGFβ and TNFα) in the presence or absence of a labeled competitor. The competitor can be a labeled monoclonal antibody raised against the target protein or a labeled native receptor of the target protein. The electrochemiluminesent binding assays were performed essentially as described in Example 5. The apparent IC50s were determined from binding data by determining the concentration of the BBI species needed to reduce the BioVeris signal by one half. BBI scaffolds without the target binding peptide in the chymotrypsin inhibitory loop were used as negative controls.

The results are summarized in Table 7 below.

TABLE 7 Binding activity of modified variant Bowman Birk Inhibitors in which the chymotrypsin loop was replaced by a VEGF-, FDF-5-, TGFβ-, or TNFα-binding variant peptide Variant Variant Resulting Modified Target Peptide Peptide Bowman Birk Inhibitor Variant Bowman BioVeris Protein Name^(•) Sequence^(▴) Scaffold Birk Inhibitor Binding* VEGF CK37281 YNLYGWT BBIt BBIt-AV of +++ (SEQ ID NO:9) SEQ ID NO:187 VEGF CK37281 YNLYGWT BBIt-A13I-A40K-F50T-V52A BBIt-AV of +++ (SEQ ID NO:9) SEQ ID NO:602 VEGF CK37281 YNLYGWT BBIt-A13I-L29P-A40K-F50T- BBIt-AV of +++ (SEQ ID NO:9) V52A SEQ ID NO:601 VEGF CK37281 YNLYGWT BBIt-A13I-S25R-M27A-L29P- BBIt-AV of +++ (SEQ ID NO:9) S31A-A40H-F50K-V52T SEQ ID NO:627 (KT8) VEGF CK37281 YNLYGWT BBIt-A13I-S25K-M27A-L29R- BBIt-AV of +++ (SEQ ID NO:9) S31E-A40K-F50Q-V52Q SEQ ID NO:628 (QQ8) VEGF CK37281 YNLYGWT BBIt-A13I-S25K-M27A-L29R- BBIt-AV of +++ (SEQ ID NO:9) S31A-A40H-F50R-V52L SEQ ID NO:630 (RL8) VEGF CK37281 YNLYGWT BBdb BBdb-AV +++ (SEQ ID NO:9) SEQ ID NO:452 VEGF CK37281 YNLYGWT BBtc BBtc-AV +++ (SEQ ID NO:9) SEQ ID NO:454 VEGF Veg K KYYLYWW BBIt BBIt-AV of +++ (SEQ ID NO:458) SEQ ID NO:640 VEGF Veg T TLWKSYW BBIt BBIt-AV of +++ (SEQ ID NO:459) SEQ ID NO:641 VEGF Veg KD KYDLYWW BBIt BBIt-AV of +++ (SEQ ID NO:460) SEQ ID NO:462 VEGF Veg KD KYDLYWW BBIt-A13I-S25K-L29P-V52K BBIt-AV of +++ (SEQ ID NO:460) SEQ ID NO:643 VEGF V1 SKHSQIT BBIt-A13I-S25R-M27A-L29P- BBIt-AV- ++ (SEQ ID NO:468) S31A-F50K-V52T V1 of SEQ ID NO:491 VEGF V2 KTNPSGS BBIt-A13I-S25R-M27A-L29P- BBIt-AV- +++ (SEQ ID NO:469) S31A-F50K-V52T V2 of SEQ ID NO:632 VEGF V3 RPTGHSL BBIt-A13I-S25R-M27A-L29P- BBIt-AV- ++ (SEQ ID NO:470) S31A-F50K-V52T V3 of SEQ ID NO:633 VEGF V4 KHSAKAE BBIt-A13I-S255-M27A-L29P- BBIt-AV- +++ (SEQ ID NO:471) S31A-F50K-V52T V4 of SEQ ID NO:634 VEGF V5 KPSSASS BBIt-A13I-S25R-M27A-L29P- BBIt-AV- ++ (SEQ ID NO:472) S31A-I40A-F50K-V52T V5 of SEQ ID NO:635 VEGF V6 PVTKRVH BBIt-A13I-S25R-M27A-L29P- BBIt-AV- +++ (SEQ ID NO:473) S31A-F50K-V52T V6 of SEQ ID NO:636 VEGF Wild-type ALSYPAQ BBI sBBI − negative (SEQ ID NO:675) SEQ ID NO:13 control for VEGF binding FGF-5 MM007 RTQPYPL BBIt-A13I-L29P-F50T-V52A MM007-Q- ++ (SEQ ID NO:670) BBIt-FGF- 5 of SEQ ID NO:432 FGF-5 FPGps2 TWIDSTP BBIt-A13I-L29P-F50T-V52A FGFps2- ++ (SEQ ID NO:671) Q-BBIt- FGF-5 of SEQ ID NO:434 FGF-5 Wild-type ALSYPAQ BBI sBBI − negative (SEQ ID NO:675) SEQ ID NO:13 control for FGF-5 binding TGFβ1 PEN3 PENINVLP BBIt-A40L-F50N PEN3-Q- ++ (SEQ ID NO:672) BBIt-TGF- A40L- F50N (SEQ ID NO:677) TGFβ1 PEN3 PENINVLP BBIt-A13I-L29P-F50T-V52A PEN3-Q- ++ (SEQ ID NO:672) BBIt-TGF (SEQ ID NO:443) TGFβ1 MM021W KHNVDWL BBIt-A13I-L29P-F50T-V52A MM0212- ++ (SEQ ID NO:673) Q-BBIt- TGF (SEQ ID NO:445) TGFβ1 WTQ WTQHIHN BBIt-A13I-L29P-F50T-V52A WTQ-Q- ++ (SEQ ID NO:674) BBIt-TGF (SEQ ID NO:447) TGFβ1 FGFps2 TWIDSTP BBIt-A13I-L29P-F50T-V52A FGFps2- − Negative (SEQ ID NO:671) Q-BBIt- control for FGF-5 of TGFβ SEQ ID NO:434 binding TNFα T1 RYWQDIP BBIt-A13I-S25R-M27A-L29P- BBIt-TNF- ++ (SEQ ID NO:474) S31A-F50K-V52T T1 SEQ ID NO:637 TNFα T2 APEPILA BBIt-A13I-S25R-M27A-L29P- BBIt-TNF- + (SEQ ID NO:475) S31A-F50K-V52T T2 SEQ ID NO:638 TNFα T3 DMINVSI BBIt-A13I-S25R-M27A-L29P- BBIt-TNF- + (SEQ ID NO:476) S31A-F50K-V52T T3 SEQ ID NO:639 TNFα V1 SKHSQIT BBIt-A13I-S25R-M27A-L29P- BBIt-AV of − Negative (SEQ ID NO:468) S31A-F50K-V52T SEQ ID NO:491 control for TNFα binding ^(•)Binding peptide used to replace the chymotrypsin loop of BBIt scaffold. ^(▴)Amino acid sequence of the binding peptide that replaces amino acids 42-48 of the BBIt of SEQ ID NO:187. *(+++) indicates an IC50 value of about 2-10 μM; (++) indicates an IC50 value of about 10-50 μM; (+) indicates an IC50 value of about 50-250 μM; and (−) indicates an IC50 value greater than about 250 μM

These data show that modified variant BBPIs in which the chymotrypsin loop is replaced by different VEGF binding peptides, and which further comprise at least one amino acid substitution in the backbone of the scaffold, specifically bind their target protein i.e. VEGF. Similarly, the modified variant BBPIs in which the chymotrypsin loop is replaced by other variant peptides e.g. FGF-5, TGFβ and TNFα, also specifically bind their corresponding target proteins i.e. FGF, TGF and TNF. In addition, other BBPI scaffolds i.e. BBtc, BBsb3 and BBdb in which the chymotrypsin loop has been replaced by a variant peptide e.g. VEGF variant peptide, are also capable of specifically binding to the VEGF target protein.

In conclusion, these data show that modified variant BBPI scaffolds comprising at least one amino acid substitution and carrying different variant peptides in place of the chymotrypsin loop retain the ability to bind the target protein that is bound by the free (ungrafted) binding peptide. Thus, the data indicate that different binding peptide sequences that were previously shown to bind their corresponding target protein can be used to replace the chymotrypsin inhibitory loop of a modified BBI protein and be expected to bind the cognate target protein.

Example 16 Identification of Peptide T-Cell Epitopes in wt BBI

The I-mune® assay was performed for the identification of peptide T-cell epitopes in wild type BBI using naïve human T-cells, as described in WO9953038A2 and Stickler et al, 2000 (J. Immunotherapy, 23(6), 654-660, [2000]). Peptides for use in the assay were prepared based on the wild-type BBI amino acid sequence DDESSKPCCDQCACTKSNPPQCRCSDMRLNSCHSACKSCICALSYPAQCFCVDITDFCYEPCKPSEDDKEN (SEQ ID NO:13). From the full length amino acid sequence of BBI, 15mer peptides were synthetically prepared by Mimotopes US West (San Diego, Calif.). Each 15mer peptide sequence was designed to overlap with the previous and subsequent 15mer, except for three residues. The 20 peptides used to screen for T-cell epitopes, corresponding to wild type BBI sequence, were:

P1 DDESSKPCCDQCACT (P1; SEQ ID NO:649) P2 SSKPCCDQCACTKSN (P2; SEQ ID NO:650) P3 PCCDQCACTKSNPPQ (P3; SEQ ID NO:651) P4 DQCACTKSNPPQCRC (P4; SEQ ID NO:652) P5 ACTKSNPPQCRCSDM (P5; SEQ ID NO:653) P6 KSNPPQCRCSDMRLN (P6; SEQ ID NO:654) P7 PPQCRCSDMRLNSCH (P7; SEQ ID NO:655) P8 CRCSDMRLNSCHSAC (P8; SEQ ID NO:656) P9 SDMRLNSCHSACKSC (P9; SEQ ID NO:657) P10 RLNSCHSACKSCICA (P10; SEQ ID NO:658) P11 SCHSACKSCICALSY (P11; SEQ ID NO:659) P12 SACKSCICALSYPAQ (P12; SEQ ID NO:660) P13 KSCICALSYPAQCFC (P13; SEQ ID NO:661) P14 ICALSYPAQCFCVDI (P14; SEQ ID NO:662) P15 LSYPAQCFCVDITDF (P15; SEQ ID NO:663) P16 PAQCFCVDITDFCYE (P16; SEQ ID NO:664) P17 CFCVDITDFCYEPCK (P17; SEQ ID NO:665) P18 VDITDFCYEPCKPSE (P18; SEQ ID NO:666) P19 TDFCYEPCKPSEDDK (P19; SEQ ID NO:667) P20 FCYEPCKPSEDDKEN (P20; SEQ ID NO:668)

Briefly, human CD4+ T cells were co-cultured with dendritic cells and peptide for the intact peptide set, using the peptides shown in Figure Y. Cytokine responses were averaged within each experiment as described in Strickler et al, 2000 and a response to a peptide was tabulated as positive if the stimulation index (SI) was greater than 2.95 (FIG. 18A). SI values for each donor were compiled for each peptide set and the percent of donors responsive to each peptide is shown in FIG. 18B. The percent background response, 2.89%, was determined as the average percent responders over all the peptides in the set. The standard deviation for this dataset was 2.21%. Major epitopes are defined as having a percentage response that is 3-fold or more than the background. Based on the data shown in FIGS. 18A and 18B, none of the peptide responses were significant based on the statistical method for an unexposed donor population with a low background response rate.

Therefore, these data indicate that the wild type BBI molecule may be considered to have low immunogenicity potential in humans.

Example 17 Personal Care Compositions

In this Example, various personal care compositions comprising any of the modified variant BBPI compounds of the present invention are provided as follows. In these formulations, the amounts are given as percentages of the total composition, unless otherwise indicated. Also, unless otherwise indicated in the following formulations, the concentration of BBI-AV (referred to as “Compound” below) ranges from about 0.01% to about 1.0%. In some formulations, the preferred concentration is in the range of about 0.1% to about 0.2%, while in other formulations, the preferred concentration is in the range of about 0.05% to about 0.1% (e.g., for some hair growth inhibition embodiments); from about 0.02 to 0.1% (e.g., for some skin lightening embodiments); from about 0.5% to about 1.0% (e.g., for some skin lightening embodiments); or at concentrations greater than about 0.1% (e.g., for some rosacea treating embodiments). Those of skill in the art know how to determine the suitable (i.e., optimum) concentration of BBI-AV for each product. In some of the compositions provided below, the “Compound” amount is indicated as “SA.” This indicates that the formulator should use the appropriate concentration of BBI-AV as indicated above herein, or as appropriate for the specific formulation.

MOISTURIZING BODYWASH (pH 7) RAW MATERIAL (INCI Designation) Amount Deionized Water QS Glycerin 4.0 PEG-6 Caprylic/Capric Glycerides 4.0 Palm Kernel Fatty acids 3.0 Sodium Laureth-3 Sulphate 45.0 Cocamide MEA 3.0 Sodium Lauroamphoacetate 25.0 Soybean Oil 10.0 Polyquaternium-10 0.70 Preservative, fragrance, color QS Compound 1000 ppm

BODY WASH RAW MATERIAL pH 8 pH 6.5 pH 7 (INCI Designation) Amount Amount Amount Deionized water QS QS QS Sodium Laureth Sulphate 12 15 8 Cocamidopropyl Betaine 8 10 15 Decyl Glucoside 0 2 1 Polyquaternium-10 0.25 0 0 Polyquaternium-7 0 0 0.7 Preservative, fragrance, color QS QS QS Compound 250 ppm 500 ppm 1000 ppm

BODY LOTION RAW MATERIAL pH 7 pH 7 pH 7.5 pH 7 (INCI Designation) Amount Amount Amount Amount Deionized Water QS QS QS QS Glycerine 8 8 0 12 Isohexadecane 3 3 3 6 Niacinamide 0 3 5 6 Isopropyl Isostearate 3 3 3 3 Polyacrylamide (and) 3 3 3 3 Isoparaffin (and) Laureth-7 Petrolatum 4 4 4 2 Nylon 12 2 2 2.5 2.5 Dimethicone 2 2 2.5 2.5 Sucrose Polycottonseed 1.5 1.5 1.5 1.5 Oil Stearyl Alcohol 97% 1 1 1 1 D Panthenol 1 1 1 1 DL-alphaTocopherol 1 1 1 1 Acetate Cetyl Alcohol 95% 0.5 0.5 0.5 1 Behenyl Alcohol 1 1 1 0.5 Cetearyl Alcohol (and) 0.4 0.4 0.5 0.5 Cetearyl Glucoside Stearic Acid 0.15 0.15 0.15 0.15 PEG-100-Stearate 0.15 0.15 0.15 0.15 Preservative, fragrance, QS QS QS QS color Compounds 250 ppm 500 ppm 750 ppm 1000 ppm

ULTRA-HIGH MOISTURIZING EMULSION RAW MATERIAL pH 7 pH 7 (INCI Designation) Amount Amount Deionized water QS QS Glycerin 12 5 PEG 400 0 10 Niacinamide 5 7 Isohexadecane 5 5 Dimethicone 3 2 Polyacrylamide (and) Isoparaffin (and) 3 3 Laureth-7 Isopropyl Isostearate 2 2 Polymethylsilsesquioxane 2 2 Cetyl Alcohol 95% 1 1 Sucrose polycottonseed oil 1 1 D-Panthenol 1 1 Tocopherol Acetate 1 1 Stearyl Alcohol 95% 0.5 0.5 Cetearyl Glucoside 0.5 0.5 Titanium dioxide 0.3 0.3 Stearic Acid 0.15 0.15 PEG-100-Stearate 0.15 0.15 Preservative, fragrance, color QS QS Compound 250 ppm 100 ppm

MOISTURIZING CREAM RAW MATERIAL pH 7 pH 7 pH 7.5 (INCI Designation) Amount Amount Amount Deionized water QS QS QS Glycerine 3 5 10 Petrolatum 3 3 0 Cetyl Alcohol 95% 1.5 1.5 1 Dimethicone Copolyol 2 2 2 Isopropyl Palmitate 1 1 0.5 Carbopol 954 (Noveon) 0.7 0.7 0.7 Dimethicone (350cs) 1 1 1 Stearyl Alcohol 97% 0.5 0.5 1 Stearic acid 0.1 0.1 0.1 Peg-100-stearate 0.1 0.1 0.1 Titanium Dioxide 0.3 0.3 0.3 Preservative, color, fragrance QS QS QS Compound 50 ppm 250 ppm 1000 ppm

FACIAL CLEANSING EMULSION RAW MATERIAL (INCI Designation) Amount Water 69.05 Disodium EDTA 0.1 Glyceryl polymethacrylate (and) Propylene glycol 1.0 Glycerin 2.0 Xanthan gum 0.5 Hydroxyethyl cellulose 0.5 Tridecyl neopentanoate 4.0 Isocetyl stearate 6.0 Octyl palmitate 8.0 Glyceryl dilaurate 4.0 PEG-20 stearate 2.0 Glyceryl stearate (and) Laureth-23 2.0 Lauryl pyrrolidone 0.5 Chamomile extract 0.2 Aloe vera (200x) 0.05 Fragrance, preservative QS Compound SA

SURFACTANT-BASED FACIAL CLEANSER RAW MATERIAL (INCI Designation) Amount Water 62.55 Acrylates/Steareth-20 methacrylate copolymer 3.3 Disodium EDTA 0.05 Glycerin 2.0 Glyceryl polymethacrylate (and) Propylene glycol (and) 0.5 PVM/MA copolymer Sodium laureth sulfate (30%) 17.5 Cetearyl alcohol 1.0 Shea butter 1.0 Disodium oleamido PEG-2 sulfosuccinate 5.0 Cocoamidopropyl Betaine 3.0 Sodium lauroyl sarcosinate 1.0 PEG-7 glyceryl cocoate 1.0 Isodecyl oleate 1.5 Peppermint extract 0.25 Eucalyptus extract 0.25 Fragrance, preservative, color, pH adjust QS Compound SA

FACIAL EXFOLIATING GEL RAW MATERIAL (INCI Designation) Amount Water 64.39 Disodium EDTA 0.05 Aloe vera (200x) 0.01 Benzophenone-4 0.25 Propylene glycol 1.0 Acrylates/C10-30 alkyl acrylate crosspolymer (2%) 20.0 Glyceryl polymethacrylate (and) Propylene glycol 10.0 Glyceryl polymethacrylate (and) Propylene glycol (and) 1.0 PVM/MA copolymer Hydrogenated jojoba oil 1.5 Fragrance, preservative, color, pH adjust QS Compound SA

FACIAL TONER RAW MATERIAL (INCI Designation) Amount Water 93.99 Disodium EDTA 0.1 Butylene glycol 2.0 Aloe vera (200x) 0.1 Allantoin 0.1 Benzophenone-4 0.5 Witch hazel extract 0.3 Propylene glycol (and) Euphrasia extract (and) 0.01 Golden seal root extract (and) Green tea extract PEG-40 hydrogenated castor oil 0.5 Quaternium-22 0.5 Sandlewood oil 0.02 Fragrance, preservative, color, pH adjust QS Compound SA

EXFOLIATING CREAM RAW MATERIAL (INCI Designation) Amount Water 68.80 Disodium EDTA 0.1 PVM/MA decadiene crosspolymer 1.0 Butylene glycol 3.0 PEG-20 stearate 1.0 Glyceryl stearate (and) Laureth-23 2.0 Diisopropyl adipate 2.0 Isodecyl oleate 2.0 Isocetyl stearoyl stearate 5.0 Myristyl myristate 1.0 Glyceryl dilaurate 2.0 Sodium hydroxide, 10% 2.6 Glyceryl polymethacrylate (and) Propylene glycol 5.0 Glyceryl polymethacrylate (and) Propylene glycol (and) 0.5 PVM/MA copolymer Hydrogenated jojoba oil 3.0 Fragrance, preservative, color, pH adjust QS Compound SA

FACIAL MASK RAW MATERIAL (INCI Designation) Amount Water 76.4 Disodium EDTA 0.1 Bentonite 12.5 Potassium C12-13 Alkyl Phosphate 5.0 Propylene glycol 4.0 Sodium Coco PG-Dimonium Chloride Phosphate 1.0 Fragrance, preservative, color, pH adjust QS Compound SA

AFTER-SHAVE BALM RAW MATERIAL (INCI Designation) Amount Water 82.12 Disodium EDTA 0.1 Acrylate copolymer 2.0 Acrylate/Stareth-20 methacrylate copolymer 1.0 Propylene glycol 3.0 Sodium hydroxide (10%) 1.28 Glyceryl stearate (and) Cetyl alcohol (and) Stearyl alcohol 3.5 (and) Behenyl alcohol (and) Palmitic acid (and) Stearic acid (and) Hydroxyethyl cetearamidopropyldimonium chloride Isocetyl stearate 1.0 C12-15 alkyl lactate 1.5 Octyldodecyl stearate 3.0 Glyceryl polymethacrylate (and) Propylene glycol (and) 1.0 PVM/MA copolymer Polyquaternium-11 0.5 Fragrance, preservative, color, pH adjust QS Compound SA

EYE GEL RAW MATERIAL (INCI Designation) Amount Water 89.14 VP/Acrylates/Lauryl methacrylate copolymer 0.5 Glycerin 5.0 Aminomethyl propanol 0.3 Aloe vera (200x) 0.05 Benzophenone-4 0.1 Glyceryl polymethacrylate (and) Propylene glycol (and) 0.2 PVM/MA copolymer Butylene glycol (and) Water (and) Witch hazel extract 0.5 Butylene glycol (and) Water (and) Cucumber extract 0.3 PEG-40 hydrogenated castor oil 0.01 Acrylates/Beheneth-25 methacrylate copolymer 2.4 Fragrance, preservative, color, pH adjust QS Compound SA

HIGH MELTING POINT LIPSTICK RAW MATERIAL (INCI Designation) Amount Ozokerite wax 5.0 Candelilla wax 11.0 Octyl dodecanol 26.0 C30-45 alkyl methicone 5.0 Cyclomethicone 4.8 Petrolatum 3.0 Lanolin oil 9.0 Avocado oil 2.0 Oleyl alcohol 8.0 Pigment/cyclomethicone 25.0 Fragrance, preservative QS Compound SA

LIPSTICK RAW MATERIAL (INCI Designation) Amount Candelilla wax 9.1 Isopropyl myristate 9.6 Lanolin 5.0 Beeswax 4.0 Paraffin (130/135) 2.0 Ozokerite wax 2.5 Castor oil 53.7 Carnauba wax 1.5 Pigments 7.5 Mineral oil 4.0 Fragrance, preservative QS Compound SA

LIP GLOSS RAW MATERIAL (INCI Designation) Amount Bis-diglyceryl polyacyladipate-1 43.5 Bis-diglyceryl polyacyladipate-2 10 Glycerol ricinoleate 10 Polyisobutene 1000 13 Lanolin wax 10 Candelilla wax 2.5 Mica (and) titanium dioxide 3 d-Panthenol 5 Fragrance, preservative, color QS Compound SA

LIP GLOSS WITH SUNSCREEN RAW MATERIAL (INCI Designation) Amount Triisostearyl Citrate 58.4 Candelilla wax 8.0 Myristyl lactate 7.5 Microcrystalline wax 5.0 Carnauba wax 2.0 Diisopropyl dimmer dilinoleate 10.0 Mica (and) Bismuth oxychloride (and) Carmine 6.0 Zinc oxide (microfine) 2.0 Fragrance, preservative QS Compound SA

LIP BALM RAW MATERIAL (INCI Designation) Amount Petrolatum 47.3 Isopropyl lanolate 6.0 Ozokerite wax 16.5 Candelilla wax 4.5 Diisopropyl dilinoleate 25.0 Retinyl palmitate 0.5 Tocopherol acetate 0.2 Fragrance, preservative QS Compound SA

WATERPROOF MASCARA RAW MATERIAL (INCI Designation) Amount Water 49.45 Propylene glycol 3.0 Triethanolamine (99%) 3.1 Acrylates/Octylacrylamine Copolymer 5.0 Diisostearoyl trimethylolpropane siloxy silicate 5.0 Candelilla wax 4.5 Beeswax 5.5 Ozokerite wax 2.0 Carnauba wax 1.0 Cetyl alcohol 3.0 Stearic acid 5.0 Iron oxides 11.0 Fragrance, preservative QS Compound SA

ANHYDROUS WATERPROOF MASCARA RAW MATERIAL (INCI Designation) Amount C9-11 Isoparaffin 30.95 Polyethylene 11.0 Candelilla wax 4.5 Hydroxylated lanolin 0.25 Pentaerythrityl rosinate 2.0 Zinc stearate 1.0 Silica silylate 1.0 Petroleum distillates (and) Quaternium-18 35.0 hectorite (and) Propylene Carbonate Iron oxides 12.0 Fragrance, preservative QS Compound SA

WATER-BASED MASCARA RAW MATERIAL (INCI Designation) Amount Water 43.32 Polyvinyl pyrrolidone (K30) 2.0 Hydroxyethyl cellulose 1.0 Triethanolamine (99%) 2.0 Disodium EDTA 0.1 Iron Oxides 10.0 Stearic acid 4.5 Glyceryl monostearate 2.0 Beeswax 7.0 Carnauba wax 4.5 Hydroxylated lanolin 1.0 Acrylates copolymer 20.0 Fragrance, preservative QS Compound SA

LIQUID EYELINER RAW MATERIAL (INCI Designation) Amount Water 50-70 Gellant 0.5-1.5 Wetting agent(s) 1-3 Polyol 4-8 Colorants 10-20 Alcohol  5-10 Film former 3-8 Fragrance, preservative QS Compound SA

NAIL ENAMEL RAW MATERIAL (INCI Designation) Amount Solvent(s) 40-70 Resin(s) 10-20 Plasticizer  3-12 Gellant 0-2 Colorants 0-3 Fragrance, preservative QS Compound SA

CUTICLE TREATMENT RAW MATERIAL (INCI Designation) Amount Petrolatum 34.8 Beeswax 7.2 Ozokerite wax 4.3 Candelilla wax 4.0 Cocoa butter 1.0 Shea butter 1.0 Glyceryl dilaurate 8.0 Ethylhexyl palmitate 20.0 C12-15 alkyl lactate 6.0 PVP/Eicosene copolymer 3.5 Diisopropyl adipate 2.0 Octinoxate 7.5 Retinyl palmitate 0.1 Tocopherol acetate 0.1 Fragrance, preservative, color, pH adjust QS Compound SA

PRESSED POWDER FORMULATIONS Pressed Eye Loose Powder Powder Foundation Blush Shadow Fillers 70-95 40-90 40-80 40-80 40-80  (e.g., talc, mica, seracite) Compression aids   0-2.5 3-5 2-5 2-7 2-10 (e.g., metallic soaps, waxes) Texture enhancers 10-40  5-40 10-40 10-40 0-30 Colorants  2-10  2-10  5-20  2-10 1-40 (e.g., iron oxides, organic colors) Pearls  0-20  0-10 0-5  0-20 0-60 (e.g. titanated mica, bismuth oxychloride) Wet binder 0-3 2-5 2-5  3-10 3-15 (e.g., Octyldodecyl stearoyl stearate, di-PPG3 myristyl ether adipate, isocetyl stearate, cetyl dimethicone) Dry binder 0-2 2-5 2-5 3-8 3-8  (e.g., calcium silicate, kaolin) Fragrance, preservative QS QS QS QS QS Compound SA SA SA SA SA

WATER-IN-OIL FOUNDATION RAW MATERIAL (INCI Designation) Amount Cyclomethicone 12.0 Dimethicone 5.0 Cyclomethicone (and) Dimethicone copolyol 20.0 Laureth-7 0.5 Colorants (hydrophobically treated) 2.2 Titanium dioxide (and) methicone 8.5 Talc (and) methicone 3.3 Water 37.2 Sodium chloride 2.0 Propylene glycol 8.0 Fragrance, preservative QS Compound SA

ANHYDROUS MAKEUP STICK RAW MATERIAL (INCI Designation) Amount Ozokerite wax 5.6 Polyethylene 5.3 Glyceryl dilaurate 5.5 Isostearyl neopentanoate 13.0 Octyldodecyl stearoyl stearate 12.0 Myristyl myristate 11.0 Ethylhexyl methoxycinnamate 7.5 PVP/Eicosene copolymer 0.5 Tocopherol acetate 0.1 Dimethicone (and) Trimethylsiloxysilicate 8.0 Cyclopentasiloxane 9.0 Mica 10.0 Talc 1.7 Titanium dioxide (and) Isopropyl titanium 8.86 triisostearate Iron oxides (and) Isopropyl titanium triisostearate 1.94 Fragrance, preservative QS Compound SA

WATER-IN-SILICONE FOUNDATION RAW MATERIAL (INCI Designation) Amount Cetyl dimethicone copolyol 0.45 Polyglycerol-4 isostearate (and) Cetyl dimethicone 1.75 copolyol (and) Hexyl laurate Polyalkylene polysiloxane copolymer 0.9 Cetyl dimethicone 0.9 Beeswax 0.7 Castor wax (and) hydrogenated castor oil 0.35 Octyl palmitate 7.0 Cyclomethicone 7.95 Phenyl trimethicone 2.2 Titanium dioxide (and) Caprylyl silane 7.5 Iron oxides (and) Caprylyl silane 1.1 Talc (and) Caprylyl silane 3.8 Cyclomethicone 7.95 Dimethicone 1.3 Water 49.55 Sodium chloride 0.5 Propylene glycol 5.3 Fragrance, preservative QS Compound SA

OIL-IN-WATER FOUNDATION RAW MATERIAL (INCI Designation) Amount Water 59.85 Polyvinylpyrrolidone 5.0 Magnesium aluminum silicate 2.0 Xanthan gum 0.4 Trisodium EDTA 0.05 Glyceryl polymethacrylate (and) Propylene glycol 1.0 (and) PVM/MA copolymer Polysorbate 20 1.0 Kaolin 0.8 Butylene glycol 4.0 Titanium dioxide 6.05 Iron oxides 1.15 Dimethicone 6.0 Ethylhexyl palmitate 2.0 PEG/PPG-25/25 Dimethicone 1.0 Tocopherol acetate 0.1 Retinyl palmitate 0.1 Silica 3.0 Cyclopentasiloxane 5.0 Fragrance, preservative QS Compound SA

SUNSCREEN FORMULAE RAW MATERIAL Amount (INCI Designation) SPF ~25 SPF ~15 Water 52.65 71.10 PVM/MA decadiene crosspolymer 0.5 0.5 Butylene glycol 3.0 3.0 Disodium EDTA 0.1 0.1 PEG-20 stearate 1.5 1.5 Glyceryl stearate (and) Laureth-23 2.0 2.0 Isostearyl neopentanoate 1.0 1.0 Ethylhexyl palmitate 2.0 2.0 Glyceryl dilaurate 0.5 0.5 Octinoxate 7.5 7.5 Oxybenzone 2.0 2.0 Ethylhexyl salicylate 3.0 3.0 Sodium hydroxide (10%) 1.3 1.3 Glyceryl polymethacrylate (and) Propylene glycol 3.0 3.0 Glyceryl polymethacrylate (and) Propylene glycol 0.5 0.5 (and) PVM/MA copolymer Styrene/Acrylates copolymer (27% solids) 18.45 — Fragrance, preservative QS QS Compound SA SA

VERY WATER-RESISTANT SUNSCREEN FORMULAE RAW MATERIAL Amount (INCI Designation) SPF ~12 SPF ~22 Water 65.16 46.53 Acrylates copolymer 3.0 3.0 Disodium EDTA 0.1 0.1 Butylene glycol 2.0 2.0 Gylceryl polymethacrylate (and) Propylene 1.0 1.0 glycol (and) PVM/MA copolymer Butylated PVP 0.05 0.05 Glyceryl stearate (and) Behenyl alcohol (and) 4.5 4.5 Palmitic acid (and) Stearic acid (and) Lecithin (and) Lauryl alcohol Tricontanyl PVP 1.0 1.0 Octyl palmitate 2.0 2.0 Octinoxate 7.5 7.5 Oxybenzone 2.0 2.0 Ethylhexyl salicylate 3.0 3.0 Tridecyl neopentanoate 3.0 3.0 Glyceryl dilaurate 0.5 0.5 Sodium hydroxide (10%) 1.89 1.89 Cyclopentasiloxane 2.0 2.0 Butylene glycol 1.0 1.0 Styrene/Acrylates copolymer (27% solids) 18.45 — Fragrance, preservative QS QS Compound SA SA

WATER-IN-SILICONE SUNSCREEN RAW MATERIAL (INCI Designation) Amount Cetyl PEG/PPG-15/15 butyl ether dimethicone 2.0 Mineral oil 3.0 Ethylhexyl palmitate 1.0 Ethylhexyl salicylate 5.0 Hydrogenated castor oil 0.5 Beeswax 0.5 Octinoxate 7.5 Polyethylene 1.0 PEG-30 dipolyhydroxystearate 2.0 Cyclopentasiloxane 5.0 Dimethicone 5.0 Sodium chloride 0.6 Acrylates/C12-22 alkylmethacrylate copolymer 0.5 Water 66.4 Fragrance, preservative QS Compound SA

LEAVE-ON HAIR CONDITIONER RAW MATERIAL (INCI Designation) Amount Deionized Water QS Isostearamidopropyl Morpholine Lactate 6.0 Hydroxyethylcellulose 1.0 Preservative, fragrance, color QS Compound 1000 ppm

CREAM RINSE (pH 4) RAW MATERIAL (INCI Designation) Amount Deionized Water QS Behentrimonium Chloride 2.0 Trilaureth-4 Phosphate 1.5 Cetyl alcohol 2.0 Citric acid QS Preservative, fragrance, color QS Compound 1000 ppm

NOURISHING HAIR CONDITIONER/TREATMENT (pH 6) RAW MATERIAL (INCI Designation) Amount Deionized Water QS Behentrimonium Methosulfate (and) Cetyl Alcohol 4.0 Wheat germ oil 1.0 Cetyl alcohol 0.5 Propylene glycol 5.0 PEG-60 Lanolin 1.0 Panthenol 2.0 Lupin amino acids 1.0 Cocodimonium Hydroxypropyl Hydrolyzed 1.0 Wheat Protein Fragrance, preservative, color QS Compound 1000 ppm

CONDITIONING SHAMPOO RAW MATERIAL (INCI Designation) Amount Deionized Water QS Sodium Laureth Sulfate 30% 27.0 Cocamidopropyl Betaine 3.7 Coco-Glucoside (and) Glyceryl Oleate 5.0 Coco-Glucoside (and) Glycol Distearate 3.0 (and) Glycerine Guar Hydroxypropyl Trimonium Chloride 0.1 Laureth-2 1.55 Fragrance, preservative, color QS Compound 1000 ppm

ANTI-DANDRUFF SHAMPOO RAW MATERIAL (INCI Designation) Amount Deionized Water QS Magnesium Aluminum Silicate 1.0 Hydroxypropyl Methylcellulose 0.8 Sodium Olefin Sulfate 40% 35.0 Lauramide DEA 4.0 Soyamide DEA 1.0 Quaternium-70 Hydrolyzed Collagen 2.0 Zinc Pyrithione 40% 4.0 Fragrance, preservative, color QS Compound 1000 ppm

CLEAR SHAMPOO RAW MATERIAL (INCI Formulations (Amounts) Designation) 1 2 3 4 5 Texapon 13.00  15.00  10.50  12.50  10.00  N 70 Dehyton 7.50 7.00 5.00 5.50 10.00  PK 45 Cetiol HE 2.00 2.50 3.50 5.00 2.30 Fragrance 0.10 0.10 0.10 0.10 0.10 Compound SA SA SA SA SA D-Panthenol 1.00 1.50 1.80 1.70 1.40 USP Preservative 0.10 0.10 0.10 0.10 0.10 Citric Acid 0.10 0.10 0.10 0.10 0.10 Luviquat 1.50 1.00 1.50 1.20 1.10 Ultra Care Sodium 1.50 1.40 1.40 1.30 1.50 Chloride Water QS (100) QS (100) QS (100) QS (100) QS (100)

SHAMPOO RAW MATERIAL (INCI Formulations (Amounts) Designation) 1 2 3 4 5 Texapon 35.00  40.00  30.00  45.00  27.00  NSO Plantacare 5.00 5.50 4.90 3.50 7.00 2000 Tego Betain 10.00  5.00 12.50  7.50 15.00  L7 Fragrance 0.10 0.10 0.10 0.10 0.10 Compound SA SA SA SA SA D-Panthenol 0.50 1.00 0.80 1.50 0.50 USP Preservative 0.10 0.10 0.10 0.10 0.10 Citric Acid 0.10 0.10 0.10 0.10 0.10 Rewopal 0.50 2.00 0.50 0.50 2.00 LA 3 Sodium 1.50 1.50 1.50 1.50 1.50 Chloride Water QS (100) QS (100) QS (100) QS (100) QS (100)

CLEAR CONDITIONING SHAMPOO RAW MATERIAL Formulations (Amounts) (INCI Designation) 1 2 3 4 5 Amphotensid GB 2009 10.00  15.00  20.00  12.00  17.00  Plantacare 2000 5.00 6.00 7.00 8.00 4.00 Tego Betain L7 15.00  12.00  10.00  18.00  20.00  Luviquat FC 550 0.30 0.20 0.20 0.20 0.30 Fragrance 0.10 0.10 0.10 0.10 0.10 Compound SA SA SA SA SA Cremophor PS 20 5.00 1.00 1.00 7.00 5.00 Preservative 0.10 0.10 0.10 0.10 0.10 Rewopal LA 3 2.00 1.00 0.50 2.00 2.00 Citric Acid 0.20 0.20 0.20 0.20 0.20 Stepan PEG 600 DS 3.00 2.00 2.00 3.00 2.50 Water QS (100) QS (100) QS (100) QS (100) QS (100)

FOAM O/W-EMULSION Formulations RAW MATERIAL (Amounts) (INCI Designation) 1 2 Stearic acid 5.00 1.00 Cetyl alcohol 5.50 Cetylstearyl alcohol 2.00 PEG-40 Stearate 8.50 PEG-20 Stearate 1.00 Caprylsäure/Caprinsäure triglyceride 4.00 2.00 C12-15 Alkylbenzoate 10.00 15.00 Cyclomethicone 4.00 Dimethicone 0.50 Compound SA SA Octylisostearate 5.00 Myristyl Myristate 2.00 Ceresin 1.50 Glycerine 3.00 Filter Hydroxypropyl distärke phosphate 1.00 3.50 BHT 0.02 Disodium EDTA 0.50 0.10 Parfüm, Konservierungsmittel QS QS Colorant QS QS Potassium hydroxide QS QS Water dem. QS (100) QS (100) pH adjusted to pH adjusted to 6.5-7.5 5.0-6.0 Emulsion 1 Emulsion 2 Gas (Stickstoff) Gas (Helium)

CONDITIONER SHAMPOO WITH PEARLESCENT Formulations RAW MATERIAL (Amounts) (INCI Designation) 1 2 3 Polyquarternium-10 0.50 0.50 0.40 Sodiumlaurethsulfat 9.00 8.50 8.90 Cocoamidopropylbetain 2.50 2.60 3.00 Benzophenon-4 1.50 0.50 1.00 Compound SA SA SA Pearlescent compound 2.00 2.50 Disodium EDTA 0.10 0.15 0.05 Preservative, Perfume, thickener QS QS QS Water dem. QS (100) QS (100) QS (100) pH adjusted to 6.0

CLEAR CONDITIONING SHAMPOO Formulations RAW MATERIAL (Amounts) (INCI Designation) 1 2 3 Polyquarternium-10 0.50 0.50 0.50 Sodiumlaurethsulfat 9.00 8.50 9.50 Compound SA SA SA Benzophenon-3 1.00 1.50 0.50 Imidosuccinicacid, Na 0.20 0.20 0.80 Preservative, Perfume, thickener QS QS QS Water dem. QS (100) QS (100) QS (100) pH adjusted to 6.0

CLEAR CONDITIONING SHAMPOO WITH VOLUME EFFECT Formulations RAW MATERIAL (Amounts) (INCI Designation) 1 2 3 Natriumlaurethsulfat 10.00 10.50 11.00 Ethylhexyl Methoxycinnamat 2.00 1.50 2.30 Compound SA SA SA Cocoamidopropylbetain 2.50 2.60 2.20 Disodium EDTA 0.01 0.10 0.01 Preservative, Perfume, thickener QS QS QS Water dem. QS (100) QS (100) QS (100) pH adjusted to 6.0

CONDITIONING SHAMPOO WITH PEARLESCENT Formulations RAW MATERIAL (Amounts) (INCI Designation) 1 2 3 Polyquarternium-10 0.50 0.50 0.40 Sodiumlaurethsulfat 9.00 8.50 8.90 Cocoamidopropylbetain 2.50 2.60 3.00 Benzophenon-4 1.50 0.50 1.00 Compound SA SA SA Pearlescent compound 2.00 2.50 Disodium EDTA 0.10 0.15 0.05 Preservative, Perfume, thickener QS QS QS Water dem. QS (100) QS (100) QS (100) pH adjusted to 6.0

CLEAR CONDITIONING SHAMPOO Formulations RAW MATERIAL (Amounts) (INCI Designation) 1 2 3 Polyquarternium-10 0.50 0.50 0.50 Sodiumlaurethsulfat 9.00 8.50 9.50 Compound SA SA SA Benzophenon-3 1.00 1.50 0.50 Imidosuccinicacid, Na 0.20 0.20 0.80 Preservative, Perfume, thickener QS QS QS Water dem. QS (100) QS (100) QS (100) pH adjusted to 6.0

CLEAR CONDITIONING SHAMPOO WITH VOLUME EFFECT Formulations RAW MATERIAL (Amounts) (INCI Designation) 1 2 3 Natriumlaurethsulfate 10.00 10.50 11.00 Ethylhexyl Methoxycinnamat 2.00 1.50 2.30 Compound SA SA SA Cocoamidopropylbetain 2.50 2.60 2.20 Disodium EDTA 0.01 0.10 0.01 Preservative, Perfume, thickener QS QS QS Water dem. QS (100) QS (100) QS (100) pH adjusted to 6.0

GEL CREME Formulations RAW MATERIAL (Amounts) (INCI Designation) 1 2 3 4 Acrylat/C10-30 Alkylacrylat Crosspolymer 0.40 0.35 0.40 0.35 Polyacrylicacid 0.20 0.22 0.20 0.22 Xanthan Gummi 0.10 0.13 0.10 0.13 Cetearylalkohol 3.00 2.50 3.00 2.50 C12-15 Alkylbenzoat 4.00 4.50 4.00 4.50 Caprylic/Capric Triglycerid 3.00 3.50 3.00 3.50 Aminobenzophenon (e.g., UVINUL A 2.00 1.50 0.75 1.00 PLUS ™) UVASorb K2A 3.00 Ethylhexyl Methoxycinnamat 3.00 1.00 Bis-Ethylhexyloxyphenol methoxyphenyl 1.50 2.00 Triazin Butyl Methoxydibenzoylmethan 2.00 Disodium Phenyl Dibenzimidazol 2.50 0.50 2.00 Tetrasulfonat Ethyhexyl Triazon 4.00 3.00 4.00 Octocrylen 4.00 Diethylhexyl Butamido Triazon 1.00 2.00 Phenylbenzimidazol Sulfonsaure 0.50 3.00 Methylen Bis-Benzotriazolyl 2.00 0.50 1.50 Tetramethylbutylphenol Ethylhexysalicylate 3.00 Drometrizol Trisiloxan 0.50 Terephthaliden Dicamphor Sulfonsäure 1.50 1.00 Diethylhexyl-2,6-naphthalate 3.50 4.00 7.00 9.00 Titanium dioxide-microfine 1.00 3.00 Zincoxide-microfine 0.25 Compound SA SA SA SA Cyclisches Dimethylpolysiloxane 5.00 5.50 5.00 5.50 Dimethicon Polydimethylsiloxane 1.00 0.60 1.00 0.60 Glycerine 1.00 1.20 1.00 1.20 Sodium hydoxide QS. QS QS QS Preservative 0.30 0.23 0.30 0.23 Perfume 0.20 0.20 Water QS (100) QS (100) QS (100) QS (100) pH adjusted to 6.0

O/W SUNSCREEN FORMULATION RAW MATERIAL Formulation (Amounts) (INCI Designations) 1 2 3 4 5 6 7 Glycerin monostearate SE 0.50 1.00 3.00 1.50 Glycerl Stearate Citrate 2.00 1.00 2.00 4.00 Stearic acid 3.00 2.00 PEG-40 Stearate 0.50 2.00 Cetyl Phosphate 1.00 Cetearyl Sulfate 0.75 Stearyl Alcohol 3.00 2.00 0.60 Cetyl Alcohol 2.50 1.10 1.50 0.60 2.00 Compound SA SA SA SA SA SA SA Aminobenzophenon (e.g., UVINUL 2.00 1.50 0.75 1.00 2.10 4.50 5.00 A PLUS ™) UVASorb K2A Ethylhexyl Methoxycinnamate 5.00 6.00 8.00 Bis-Ethylhexyloxyphenol 1.50 2.00 2.50 2.50 methoxyphenyl Triazin Butyl Methoxydibenzoylmethane 2.00 2.00 1.50 Dinatrium Phenyl Dibenzimidazol 2.50 0.50 2.00 0.30 Tetrasulfonate Ethyhexyl Triazone 4.00 3.00 4.00 2.00 Octocrylen 4.00 7.50 Diethylhexyl Butamido Triazon 1.00 2.00 1.00 1.00 Phenylbenzimidazol Sulfonsäure 0.50 3.00 Methylen Bis-Benzotriazolyl 2.00 0.50 1.50 2.50 Tetramethylbutylphenol Ethylhexysalicylat 3.00 5.00 Drometrizol Trisiloxan 0.50 1.00 Terephthaliden Dicamphor Sulfonic 1.50 1.00 1.00 0.50 Acid Diethylhexyl-2,6-naphthalat 3.50 7.00 6.00 9.00 Titandioxid-microfine 1.00 3.00 3.50 1.50 Zinkoxid-microfine 0.25 2.00 C12-15 Alkyl Benzoate 0.25 4.00 7.00 Dicapryl Ether 3.50 2.00 Butylenglycol Dicaprylat/Dicaprat 5.00 6.00 Cocoglyceride 6.00 2.00 Dimethicon 0.50 1.00 2.00 Cyclomethicone 2.00 0.50 0.50 Shea Butter 2.00 PVP Hexadecen Copolymer 0.20 0.50 1.00 Glycerin 3.00 7.50 7.50 5.00 2.50 Xanthan Gum 0.15 0.05 0.30 Sodium Carbomer 0.20 0.15 0.25 Vitamin E Acetat 0.60 0.23 0.70 1.00 Fucogel 1000 3.00 10.00  Glycin Soja 0.50 1.50 1.00 Ethylhexyloxyglycin 0.30 DMDM Hydantoin 0.60 0.40 0.20 Glyacil-L 0.18 0.20 Methylparaben 0.15 0.25 0.50 Phenoxyethanol 1.00 0.40 0.40 0.50 0.40 Trinatrium EDTA 0.02 0.05 Iminosuccinicacid 0.25 1.00 Ethanol 2.00 1.50 3.00 1.20 5.00 Perfume 0.10 0.25 0.30 0.40 0.20 Water QS (100) QS (100) QS (100) QS (100) QS (100) QS (100) QS (100)

O/W SUNSCREEN FORMULATION RAW MATERIAL Formulation (Amounts) (INCI Designations) 1 2 3 4 5 6 7 Glycerin monostearate SE 0.50 1.00 3.00 1.50 Glycerl Stearate Citrate 2.00 1.00 2.00 4.00 Stearic acid 3.00 2.00 PEG-40 Stearate 0.50 2.00 Cetyl Phosphate 1.00 Cetearyl Sulfate 0.75 Stearyl Alcohol 3.00 2.00 0.60 Cetyl Alcohol 2.50 1.10 1.50 0.60 2.00 Compound SA SA SA SA SA SA SA Aminobenzophenon (e.g., UVINUL 2.00 1.50 0.75 1.00 2.10 4.50 5.00 A PLUS ™) UVASorb K2A Ethylhexyl Methoxycinnamate 5.00 6.00 8.00 Bis-Ethylhexyloxyphenol 1.50 2.00 2.50 2.50 methoxyphenyl Triazin Butyl Methoxydibenzoylmethane 2.00 2.00 1.50 Dinatrium Phenyl Dibenzimidazol 2.50 0.50 2.00 0.30 Tetrasulfonate Ethyhexyl Triazone 4.00 3.00 4.00 2.00 Octocrylen 4.00 7.50 Diethylhexyl Butamido Triazon 1.00 2.00 1.00 1.00 Phenylbenzimidazol Sulfonsäure 0.50 3.00 Methylen Bis-Benzotriazolyl 2.00 0.50 1.50 2.50 Tetramethylbutylphenol Ethylhexysalicylat 3.00 5.00 Drometrizol Trisiloxan 0.50 1.00 Terephthaliden Dicamphor Sulfonic 1.50 1.00 1.00 0.50 Acid Diethylhexyl-2,6-naphthalat 3.50 7.00 6.00 9.00 Titandioxid-microfine 1.00 3.00 3.50 1.50 Zinkoxid-microfine 0.25 2.00 C12-15 Alkyl Benzoate 0.25 4.00 7.00 Dicapryl Ether 3.50 2.00 Butylenglycol Dicaprylat/Dicaprat 5.00 6.00 Cocoglyceride 6.00 2.00 Dimethicon 0.50 1.00 2.00 Cyclomethicone 2.00 0.50 0.50 Shea Butter 2.00 PVP Hexadecen Copolymer 0.20 0.50 1.00 Glycerin 3.00 7.50 7.50 5.00 2.50 Xanthan Gum 0.15 0.05 0.30 Sodium Carbomer 0.20 0.15 0.25 Vitamin E Acetat 0.60 0.23 0.70 1.00 Fucogel 1000 3.00 10.00  Glycin Soja 0.50 1.50 1.00 Ethylhexyloxyglycin 0.30 DMDM Hydantoin 0.60 0.40 0.20 Glyacil-L 0.18 0.20 Methylparaben 0.15 0.25 0.50 Phenoxyethanol 1.00 0.40 0.40 0.50 0.40 Trinatrium EDTA 0.02 0.05 Iminosuccinicacid 0.25 1.00 Ethanol 2.00 1.50 3.00 1.20 5.00 Perfume 0.10 0.25 0.30 0.40 0.20 Water QS (100) QS (100) QS (100) QS (100) QS (100) QS (100) QS (100)

HYDRODISPERSION RAW MATERIAL Formulations (Amounts) (INCI Designation) 1 2 3 4 5 Ceteaereth-20 1.00 0.50 Cetyl Alkohol 1.00 Sodium Carbomer 0.20 0.30 Acrylat/C10-30 Alkyl Acrylat 0.50 0.40 0.10 0.50 Crosspolymer Xanthan Gummi 0.30 0.15 Compound SA SA SA SA SA Aminobenzophenon (e.g., UVINUL 2.00 1.50 0.75 1.00 2.10 A PLUS ™) UVASorb K2A 3.50 Ethylhexyl Methoxycinnamat 5.00 Bis-Ethylhexyloxyphenol 1.50 2.00 2.50 methoxyphenyl Triazin Butyl Methoxydibenzoylmethan 2.00 2.00 Dinatrium Phenyl Dibenzimidazol 2.50 0.50 2.00 Tetrasulfonat Ethyhexyl Triazon 4.00 3.00 4.00 Octocrylen 4.00 Diethylhexyl Butamido Triazon 1.00 2.00 1.00 Phenylbenzimidazol Sulfonsäure 0.50 3.00 Methylen Bis-Benzotriazolyl 2.00 0.50 1.50 2.50 Tetramethylbutylphenol Ethylhexysalicylat 3.00 Drometrizol Trisiloxan 0.50 Terephthaliden Dicamphor 1.50 1.00 1.00 Sulfonsäure Diethylhexyl-2,6-naphthalat 7.00 9.00 Titaniumdioxide-microfine 1.00 3.00 3.50 Zincoxide-microfine 0.25 C12-15 Alkyl Benzoat 2.00 2.50 Dicapryl Ether 4.00 Butylenglycol Dicaprylat/Dicaprat 4.00 2.00 6.00 Dicapryl Carbonat 2.00 6.00 Dimethicon 0.50 1.00 Phenyltrimethicon 2.00 0.50 Shea Butter 2.00 5.00 PVP Hexadecen Copolymer 0.50 0.50 1.00 Tricontanyl PVP 0.50 1.00 Ethylhexylglycerin 1.00 0.80 Glycerin 3.00 7.50 7.50 8.50 Gylcin Soja 1.50 1.00 Vitamin E Acetat 0.50 0.25 1.00 Alpha-Glucosilrutin 0.60 0.25 Fucogel 1000 2.50 0.50 2.00 DMDM Hydantoin 0.60 0.45 0.25 Glyacil-S 0.20 Methylparaben 0.50 0.25 0.15 Phenoxyethanol 0.50 0.40 1.00 Trinatrium EDTA 0.01 0.05 0.10 Ethanol 3.00 2.00 1.50 7.00 Perfume 0.20 0.05 0.40 Water QS (100) QS (100) QS (100) QS (100) QS (100)

W/O SUNSCREEN EMULSION RAW MATERIAL Formulations (Amounts) (INCI Designation) 1 2 3 4 5 Cetyldimethicon Copolyol 2.50 4.00 Polyglyceryl-2-dipolyhydroxystearat 5.00 4.50 PEG-30-dipolyhydroxystearat 5.00 Compound SA SA SA SA SA Aminobenzophenon (e.g., 2.00 1.50 0.75 1.00 2.10 UVINUL A PLUS ™) UVASorb K2A 2.00 Ethylhexyl Methoxycinnamat 5.00 Bis-Ethylhexyloxyphenol 1.50 2.00 2.50 methoxyphenyl Triazin Butyl Methoxydibenzoylmethan 2.00 2.00 Dinatrium Phenyl Dibenzimidazol 2.50 0.50 2.00 Tetrasulfonat Ethyhexyl Triazon 4.00 3.00 4.00 Octocrylen 4.00 Diethylhexyl Butamido Triazon 1.00 2.00 1.00 Phenylbenzimidazol Sulfonsäure 0.50 3.00 Methylen Bis-Benzotriazolyl 2.00 0.50 1.50 2.50 Tetramethylbutylphenol Ethylhexysalicylat 3.00 Drometrizol Trisiloxan 0.50 Terephthaliden Dicamphor 1.50 1.00 1.00 Sulfonsäure Diethylhexyl-2,6-naphthalat 7.00 4.00 Titaniumdioxide-microfine 1.00 3.00 3.50 Zincoxide-microfine 0.25 Mineraloil 12.00 10.00 8.00 C12-15 Alkyl Benzoat 9.00 Dicaprylyl Ether 10.00 7.00 Butylenglycol Dicaprylat/Dicaprat 2.00 8.00 4.00 Dicaprylyl Carbonat 5.00 6.00 Dimethicon 4.00 1.00 5.00 Cyclomethicon 2.00 2.50 2.00 Shea Butter 3.00 Vaseline 4.50 PVP Hexadecen Copolymer 0.50 0.50 1.00 Ethylhexylglycerin 0.30 1.00 0.50 Glycerin 3.00 7.50 7.50 8.50 Glycin Soja 1.00 1.50 1.00 MgSO4 1.00 0.50 0.50 MgCl2 1.00 0.70 Vitamin E Acetat 0.50 0.25 1.00 Ascorbyl Palmitat 0.50 2.00 Fucogel 1000 3.50 1.00 DMDM Hydantoin 0.60 0.40 0.20 Methylparaben 0.50 0.25 0.15 Phenoxyethanol 0.50 0.40 1.00 Trisodium EDTA 0.12 0.05 0.30 Ethanol 3.00 1.50 5.00 Perfume 0.20 0.40 0.35 Water QS (100) QS (100) QS (100) QS (100) QS (100)

STICKS Formulations RAW MATERIAL (Amounts) (INCI Designation) 1 2 3 4 Caprylic/Capric Triglycerid 12.00  10.00  6.00 Octyldodecanol 7.00 14.00  8.00 3.00 Butylene Glycol Dicaprylat/Dicaprat 12.00  Pentaerythrityl Tetraisostearat 10.00  6.00 8.00 7.00 Polyglyceryl-3 Diisostearat 2.50 Bis-Diglyceryl Polyacyladipate-2 9.00 8.00 10.00  8.00 Cetearyl Alcohol 8.00 11.00  9.00 7.00 Myristyl Myristate 3.50 3.00 4.00 3.00 Beeswax 5.00 5.00 6.00 6.00 Cera Carnauba 1.50 2.00 2.00 1.50 Cera Alba 0.50 0.50 0.50 0.40 C16-40 Alkyl Stearat 1.50 1.50 1.50 Compound SA SA SA SA Aminobenzophenon (e.g., UVINUL A 2.00 1.50 0.75 9.00 PLUS ™) UVASorb K2A 2.00 4.00 Ethylhexyl Methoxycinnamat 3.00 Bis-Ethylhexyloxyphenol methoxyphenyl 1.50 2.00 Triazin Butyl Methoxydibenzoylmethan 2.00 Dinatrium Phenyl Dibenzimidazol 2.50 0.50 2.00 Tetrasulfonat Ethyhexyl Triazon 4.00 3.00 4.00 Octocrylen 4.00 Diethylhexyl Butamido Triazon 1.00 2.00 Phenylbenzimidazol Sulfonsaure 0.50 3.00 Methylen Bis-Benzotriazolyl 2.00 0.50 1.50 Tetramethylbutylphenol Ethylhexysalicylat 3.00 Drometrizol Trisiloxan 0.50 Terephthaliden Dicamphor Sulfonsäure 1.50 1.00 Diethylhexyl-2,6-naphthalat 7.00 Titaniumdioxide-microfine 1.00 3.00 Zincoxide-microfine 0.25 Vitamin E Acetat 0.50 1.00 Ascorbyl Palmitat 0.05 0.05 Buxux Chinensis 2.00 1.00 1.00 Perfume, BHT 0.10 0.25 0,35 Ricinus Communis QS (100) QS (100) QS (100) QS (100)

PIT-EMULSION RAW MATERIAL Formulations (Amounts) (INCI Designation) 1 2 3 4 5 6 7 8 Glycerinmonostearat SE 0.50 2.00 3.00 5.00 0.50 4.00 Glyceryl Isostearat 3.50 4.00 2.00 Isoceteth-20 0.50 2.00 Ceteareth-12 5.00 1.00 3.50 5.00 Ceteareth-20 5.00 1.00 3.50 PEG-100 Stearat 2.80 2.30 3.30 Cetyl Alkohol 5.20 1.20 1.00 1.30 0.50 0.30 Cetyl Palmitat 2.50 1.20 1.50 0.50 1.50 Cetyl Dimethicon Copolyol 0.50 1.00 Polyglyceryl-2 0.75 0.30 Compound SA SA SA SA SA SA SA SA Aminobenzophenon (e.g., UVINUL 2.00 1.50 0.75 1.00 2.10 4.50 5.00 2.10 A PLUS ™) UVASorb K2A 4.00 1.50 Ethylhexyl Methoxycinnamat 5.00 6.00 8.00 5.00 Bis-Ethylhexyloxyphenol 1.50 2.00 2.50 2.50 2.50 methoxyphenyl Triazin Butyl Methoxydibenzoylmethan 2.00 2.00 1.50 2.00 Dinatrium Phenyl Dibenzimidazol 2.50 0.50 2.00 0.30 Tetrasulfonat Ethyhexyl Triazon 4.00 3.00 4.00 2.00 Octocrylen 4.00 7.50 Diethylhexyl Butamido Triazon 1.00 2.00 1.00 1.00 1.00 Phenylbenzimidazol Sulfonsäure 0.50 3.00 Methylen Bis-Benzotriazolyl 2.00 0.50 1.50 2.50 2.50 Tetramethylbutylphenol Ethylhexysalicylat 3.00 5.00 Drometrizol Trisiloxan 0.50 1.00 Terephthaliden Dicamphor 1.50 1.00 1.00 0.50 1.00 Sulfonsäure Diethylhexyl-2,6-naphthalat 7.00 10.00  7.50 8.00 Titandioxid-microfine 1.00 3.00 3.50 1.50 3.50 Zinkoxid-microfine 0.25 2.00 C12-15 Alkyl Benzoat 3.50 6.35 0.10 Cocoglyceride 3.00 3.00 1.00 Dicapryl Ether 4.50 Dicaprylyl Carbonat 4.30 3.00 7.00 Dibutyl Adipate 0.50 0.30 Phenyltrimethicone 2.00 3.50 2.00 Cyclomethicon 3.00 Ethyl Galaktomannan 0.50 2.00 Hydrierte Coco-Glyceride 3.00 4.00 Abil Wax 2440 1.50 2.00 PVP Hexadecen Copolymer 1.00 1.20 Glycerin 4.00 6.00 5.00 8.00 10.00  Vitamin E Acetat 0.20 0.30 0.40 0.30 Shea Butter 2.00 3.60 2.00 Iodopropyl Butylcarbamat 0.12 0.20 Fucogel 1000 0.10 DMDM Hydantoin 0.10 0.12 0.13 Methylparaben 0.50 0.30 0.35 Phenoxyethanol 0.50 0.40 1.00 Octoxyglycerin 0.30 1.00 0.35 Ethanol 2.00 2.00 5.00 Trinatrium EDTA 0.40 0.15 0.20 Perfume 0.20 0.20 0.24 0.16 0.10 0.10 Water QS (100) QS (100) QS (100) QS (100) QS (100) QS (100) QS (100) QS (100)

GEL CREME Formulations RAW MATERIAL (Amounts) (INCI Designation) 1 2 3 4 Acrylat/C10-30 0.40 0.35 0.40 0.35 alkylacrylat crosspolymer Polyacrylic acid 0.20 0.22 0.20 0.22 Luvigel EM 1.50 2.50 2.80 3.50 Xanthan gum 0.10 0.13 0.10 0.13 Cetearylalkohol 3.00 2.50 3.00 2.50 C12-15 Alkylbenzoate 4.00 4.50 4.00 4.50 Caprylic/Capric 3.00 3.50 3.00 3.50 Triglyceride Titan dioxide-microfine 1.00 1.50 Zinc oxide-microfine 2.00 0.25 Compound SA SA SA SA Dihydroxyacetone 3.00 5.00 Cyclisches 5.00 5.50 5.00 5.50 Dimethylpolysiloxan Dimethicon 1.00 0.60 1.00 0.60 Polydimethylsiloxan Glycerine 1.00 1.20 1.00 1.20 Natrium hydroxide QS QS QS QS Preservatives 0.30 0.23 0.30 0.23 Perfume 0.20 0.20 Water QS (100) QS (100) QS (100) QS (100) pH adjusted to 6.0

O/W SELF TANNER FORMULATIONS RAW MATERIAL Formulations (Amounts) (INCI Designation) 1 2 3 4 5 6 7 Glycerin monostearate 0.50 1.00 3.00 1.50 SE Glycerlstearate citrate 2.00 1.00 2.00 4.00 Stearic acid 3.00 2.00 PEG-40 stearate 0.50 2.00 Cetyl phosphate 1.00 Cetearyl sulfate 0.75 Stearyl alcohol 3.00 2.00 0.60 Cetyl alcohol 2.50 1.10 1.50 0.60 2.00 Compound SA SA SA SA SA SA SA Dihydroxy acetone 3.00 5.00 4   Titanium dioxide- 1.00 1.50 1.50 microfine Zinc oxide-microfine 0.25 2.00 C12-15 Alkyl benzoate 0.25 4.00 7.00 Dicapryl ether 3.50 2.00 Butylenglycol 5.00 6.00 Dicaprylaet/Dicaprat Cocoglyceride 6.00 2.00 Dimethicon 0.50 1.00 2.00 Cyclomethicon 2.00 0.50 0.50 Shea butter 2.00 PVP hexadecen 0.20 0.50 1.00 copolymer Glycerin 3.00 7.50 7.50 5.00 2.50 Xanthan gum 0.15 0.05 0.30 Sodium carbomer 0.20 0.15 0.25 Vitamin E acetate 0.60 0.23 0.70 1.00 Fucogel 1000 3.00 10.00 Glycin Soja 0.50 1.50 1.00 Ethylhexyloxy glycin 0.30 DMDM hydantoin 0.60 0.40 0.20 Glyacil-L 0.18 0.20 Methylparaben 0.15 0.25 0.50 Phenoxyethanol 1.00 0.40 0.40 0.50 0.40 Trinatrium EDTA 0.02 0.05 Iminobernsteinsäure 0.25 1.00 Ethanol 2.00 1.50 3.00 1.20 5.00 Perfume 0.10 0.25 0.30 0.40 0.20 Water QS (100) QS (100) QS (100) QS (100) QS (100) QS (100) QS (100)

O/W MAKE UP RAW MATERIAL Formulations (Amounts) (INCI Desigation) 1 2 3 4 5 6 7 Glycerinmonostearat 0.50 1.00 3.00 1.50 SE Glycerl Stearat Citrat 2.00 1.00 2.00 4.00 Stearicacid 3.00 2.00 PEG-40 Stearat 0.50 2.00 Cetyl Phosphat 1.00 Cetearyl Sulfat 0.75 Stearyl Alkohol 3.00 2.00 0.60 Cetyl Alkohol 2.50 1.10 1.50 0.60 2.00 Compound SA SA SA SA SA SA SA Titaniumoxide 10.00 12.00 9.00 8.50 11.00 9.50 10.00 Ironoxide 2.00 4.00 3.00 5.00 3.40 6.00 4.40 Zincoxide 4.00 2.00 3.00 C12-15 Alkyl Benzoat 0.25 4.00 7.00 Dicapryl Ether 3.50 2.00 Butylenglycol 5.00 6.00 Dicaprylat/Dicaprat Cocoglyceride 6.00 2.00 Dimethicon 0.50 1.00 2.00 Cyclomethicon 2.00 0.50 0.50 Shea Butter 2.00 PVP Hexadecen 0.20 0.50 1.00 Copolymer Glycerin 3.00 7.50 7.50 5.00 2.50 Xanthan Gummi 0.15 0.05 0.30 Sodium Carbomer 0.20 0.15 0.25 Vitamin E Acetat 0.60 0.23 0.70 1.00 Glycin Soja 0.50 1.50 1.00 Ethylhexyloxyglycin 0.30 DMDM Hydantoin 0.60 0.40 0.20 Glyacil-L 0.18 0.20 Methylparaben 0.15 0.25 0.50 Phenoxyethanol 1.00 0.40 0.40 0.50 0.40 Trinatrium EDTA 0.02 0.05 Iminosuccinicacid 0.25 1.00 Ethanol 2.00 1.50 3.00 1.20 5.00 Perfume 0.10 0.25 0.30 0.40 0.20 Water QS (100) QS (100) QS (100) QS (100) QS (100) QS (100) QS (100)

SELF TANNER HYDRODISPERSION RAW MATERIAL Formulations (Amounts) (INCI Designation) 1 2 3 4 5 Ceteaereth-20 1.00 0.50 Cetyl Alkohol 1.00 Luvigel EM 2.00 2.50 2.00 Acrylat/C10-30 Alkyl Acrylat 0.50 0.40 0.10 0.50 Crosspolymer Xanthan Gummi 0.30 0.15 Compound SA SA SA SA SA Dihydroxyaceton 3.00 5.00 Aminobenzophenon (e.g., UVINUL 2.00 1.50 0.75 1.00 2.10 A PLUS ™) Titandioxid-microfine 1.00 1.00 1.00 Zinkoxid-microfine 1.90 0.25 C12-15 Alkyl Benzoat 2.00 2.50 Dicapryl Ether 4.00 Butylenglycol Dicaprylat/Dicaprat 4.00 2.00 6.00 Dicapryl Carbonat 2.00 6.00 Dimethicon 0.50 1.00 Phenyltrimethicon 2.00 0.50 Shea Butter 2.00 5.00 PVP Hexadecen Copolymer 0.50 0.50 1.00 Tricontanyl PVP 0.50 1.00 Ethylhexylglycerin 1.00 0.80 Glycerin 3.00 7.50 7.50 8.50 Gylcin Soja 1.50 1.00 Vitamin E Acetat 0.50 0.25 1.00 Alpha-Glucosilrutin 0.60 0.25 DMDM Hydantoin 0.60 0.45 0.25 Glyacil-S 0.20 Methylparaben 0.50 0.25 0.15 Phenoxyethanol 0.50 0.40 1.00 Trinatrium EDTA 0.01 0.05 0.10 Ethanol 3.00 2.00 1.50 7.00 Parfüm 0.20 0.05 0.40 Water QS (100) QS (100) QS (100) QS (100) QS (100)

AFTER SUN HYDRODISPERSION Formulations (Amounts) 1 2 3 4 5 Ceteaereth-20 1.00 0.50 Cetyl Alkohol 1.00 Luvigel EM 2.00 2.50 2.00 Acrylat/C10-30 Alkyl Acrylat 0.50 0.30 0.40 0.10 0.50 Crosspolymer Xanthan Gummi 0.30 0.15 Compound SA SA SA SA SA C12-15 Alkyl Benzoat 2.00 2.50 Dicapryl Ether 4.00 Butylenglycol 4.00 2.00 6.00 Dicaprylat/Dicaprat Dicapryl Carbonat 2.00 6.00 Dimethicon 0.50 1.00 Phenyltrimethicon 2.00 0.50 Tricontanyl PVP 0.50 1.00 Ethylhexylglycerin 1.00 0.80 Glycerin 3.00 7.50 7.50 8.50 Gylcin Soja 1.50 1.00 Vitamin E Acetat 0.50 0.25 1.00 Alpha-Glucosilrutin 0.60 0.25 Trinatrium EDTA 0.01 0.05 0.10 Ethanol 1.00 10.00  8.00 12.00  9.00 Perfume 0.20 0.05 0.40 Water QS (100) QS (100) QS (100) QS (100) QS (100)

WO-EMULSIONS RAW MATERIAL Formulations (Amounts) (INCI Designation) 1 2 3 4 5 Cetyldimethicon Copolyol 2.50 4.00 Polyglyceryl-2-dipolyhydroxystearat 5.00 4.50 PEG-30-dipolyhydroxystearat 5.00 Compound SA SA SA SA SA Aminobenzophenon (e.g., UVINUL 2.00 1.50 0.75 1.00 2.10 A PLUS ™) Titaniumdioxide-microfine 1.00 3.00 3.50 Zincoxide-microfine 0.90 0.25 Mineralöl 12.00 10.00 8.00 C12-15 Alkyl Benzoat 9.00 Dicaprylyl Ether 10.00 7.00 Butylenglycol Dicaprylat/Dicaprat 2.00 8.00 4.00 Dicaprylyl Carbonat 5.00 6.00 Dimethicon 4.00 1.00 5.00 Cyclomethicon 2.00 25.00 2.00 Shea Butter 3.00 Vaseline 4.50 PVP Hexadecen Copolymer 0.50 0.50 1.00 Ethylhexylglycerin 0.30 1.00 0.50 Glycerin 3.00 7.50 7.50 8.50 Glycin Soja 1.00 1.50 1.00 MgSO4 1.00 0.50 0.50 MgCl2 1.00 0.70 Vitamin E Acetat 0.50 0.25 1.00 Ascorbyl Palmitat 0.50 2.00 Fucogel 1000 3.50 7.00 DMDM Hydantoin 0.60 0.40 0.20 Methylparaben 0.50 0.25 0.15 Phenoxyethanol 0.50 0.40 1.00 Trinatrium EDTA 0.12 0.05 0.30 Ethanol 3.00 1.50 5.00 Perfume 0.20 0.40 0.35 Water QS (100) QS (100) QS (100) QS (100) QS (100)

SOLID STABILIZED EMULSIONS (PICKERING EMULSIONS) RAW MATERIAL Formulations (Amounts) (INCI Designation) 1 2 3 4 5 Mineral oil 16.00 16.00 Octyldodecanol 9.00 9.00 5.00 Caprylic/Capric Triglycerid 9.00 9.00 6.00 C12-15 Alkyl Benzoat 5.00 8.00 Butylen Glycol Dicaprylat/Dicaprat 8.00 Dicaprylyl Ether 9.00 4.00 Dicaprylyl Carbonat 9.00 Hydroxyoctacosanyl Hydroxystearat 2.00 2.00 2.20 2.50 1.50 Disteardimonium Hectorit 1.00 0.75 0.50 0.25 Cera Microcristallina + Paraffinum 0.35 5.00 Liquidum Hydroxypropyl Methylcellulose 0.10 0.05 Dimethicon 3.00 Compound SA SA SA SA SA Titaniumdioxide + Alumina + 3.00 Simethicon + Aqua Titaniumdioxide + 2.00 4.00 2.00 4.00 Trimethoxycaprylylsilan Silica Dimethyl Silylat 2.50 6.00 2.50 Bornitrid 1.00 Stärke/-Natriummetaphosphat- 2.00 Polymer Tapioca Stärke 0.50 Sodium Chlorid 5.00 7.00 8.50 3.00 4.50 Glycerin 1.00 Trinatrium EDTA 1.00 1.00 1.00 1.00 1.00 Vitamin E Acetat 5.00 10.00 3.00 6.00 10.00 Ascorbyl Palmitat 1.00 1.00 1.00 Methylparaben 0.60 0.20 Propylparaben 0.20 Phenoxyethanol 0.20 Hexamidin Diisethionat 0.40 0.50 0.40 Diazolidinyl Harnstoff 0.08 Ethanol 0.23 0.20 Perfume 5.00 3.00 4.00 Water 0.20 0.30 0.10 QS (100) QS (100) QS (100) QS (100) QS (100)

STICKS Formulations RAW MATERIAL (Amounts) (INCI Designation) 1 2 3 4 Caprylic/Capric Triglycerid 12.00  10.00  6.00 Octyldodecanol 7.00 14.00  8.00 3.00 Butylene Glycol Dicaprylat/Dicaprat 12.00  Pentaerythrityl Tetraisostearat 10.00  6.00 8.00 7.00 Polyglyceryl-3 Diisostearat 2.50 Bis-Diglyceryl Polyacyladipate-2 9.00 8.00 10.00  8.00 Cetearyl Alcohol 8.00 11.00  9.00 7.00 Myristyl Myristate 3.50 3.00 4.00 3.00 Beeswax 5.00 5.00 6.00 6.00 Cera Carnauba 1.50 2.00 2.00 1.50 Cera Alba 0.50 0.50 0.50 0.40 C16-40 Alkyl Stearat 1.50 1.50 1.50 Compound SA SA SA SA Aminobenzophenon (e.g., UVINUL A 2.00 1.50 0.75 9.00 PLUS ™) Titaniumdioxide-microfine 1.00 3.00 Zincoxide-microfine 1.00 0.25 Vitamin E Acetat 0.50 1.00 Ascorbyl Palmitat 0.05 0.05 Buxux Chinensis 2.00 1.00 1.00 Perfume, BHT 0.10 0.25 0.35 Ricinus Communis QS (100) QS (100) QS (100) QS (100)

SELF TANNER PIT-EMULSION Formulations (Amounts) 1 2 3 4 5 6 7 8 Glycerinmonostearat SE 0.50 2.00 3.00 5.00 0.50 4.00 Glyceryl Isostearat 3.50 4.00 2.00 Isoceteth-20 0.50 2.00 Ceteareth-12 5.00 1.00 3.50 5.00 Ceteareth-20 5.00 1.00 3.50 PEG-100 Stearat 2.80 2.30 3.30 Cetyl Alkohol 5.20 1.20 1.00 1.30 0.50 0.30 Cetyl Palmitat 2.50 1.20 1.50 0.50 1.50 Cetyl Dimethicon Copolyol 0.50 1.00 Polyglyceryl-2 0.75 0.30 Compound SA SA SA SA SA SA SA SA Dihydroxyaceton 3.00 5.00 4.00 Aminobenzophenon (e.g., UVINUL 2.00 1.50 0.75 1.00 2.10 4.50 5.00 2.10 A PLUS ™) Titandioxide-microfine 1.00 1.50 3.50 1.50 1.00 Zinkoxide-microfine 1.00 0.25 2.00 1.50 C12-15 Alkyl Benzoat 3.50 6.35 0.10 Cocoglyceride 3.00 3.00 1.00 Dicapryl Ether 4.50 Dicaprylyl Carbonat 4.30 3.00 7.00 Dibutyl Adipate 0.50 0.30 Phenyltrimethicone 2.00 3.50 2.00 Cyclomethicon 3.00 Ethyl Galaktomannan 0.50 2.00 Hydrogenated CoCo-Glyceride 3.00 4.00 Abil Wax 2440 1.50 2.00 PVP Hexadecen Copolymer 1.00 1.20 Glycerin 4.00 6.00 5.00 8.00 10.00  Vitamin E Acetat 0.20 0.30 0.40 0.30 Shea Butter 2.00 3.60 2.00 Iodopropyl Butylcarbamat 0.12 0.20 DMDM Hydantoin 0.10 0.12 0.13 Methylparaben 0.50 0.30 0.35 Phenoxyethanol 0.50 0.40 1.00 Octoxyglycerin 0.30 1.00 0.35 Ethanol 2.00 2.00 5.00 Trinatrium EDTA 0.40 0.15 0.20 Perfume 0.20 0.20 0.24 0.16 0.10 0.10 Water QS (100) QS (100) QS (100) QS (100) QS (100) QS (100) QS (100) QS (100)

OILGELS Formulations RAW MATERIAL (Amounts) (INCI Designation) 1 2 3 4 Caprylic/Capric 12.00 10.00 6.00 Triglycerid Octyldodecanol 7.00 14.00 8.00 3.00 Butylene Glycol 12.00 Dicaprylat/Dicaprat Pentaerythrityl 10.00 6.00 8.00 7.00 Tetraisostearat Polyglyceryl-3 2.50 Diisostearat Bis-Diglyceryl 9.00 8.00 10.00 8.00 Polyacyladipate-2 Myristyl Myristate 3.50 3.00 4.00 3.00 Bentone-34 5.00 5.00 6.00 6.00 Propylencarbonat 15.00 20.00 18.00 19.50 Compound SA SA SA SA Vitamin E Acetat 0.50 1.00 Ascorbyl Palmitat 0.05 0.05 Buxux Chinensis 2.00 1.00 1.00 Perfume, BHT 0.10 0.25 0.35 Ricinus Communis QS (100) QS (100) QS (100) QS (100)

In still further embodiments, the present invention comprises at least one inorganic pigment. In some preferred embodiments, these inorganic pigments are based on metaloxides and/or other water slightly soluble or insoluble metal compounds, including but not limited to compounds such as zinc oxides (ZnO), titanium (TiO₂), iron (e.g., Fe₂O₃), zirconium (ZrO₂), silica (SiO₂), manganese (e.g., MnO), aluminium (Al₂O₃), cer (e.g., Ce₂O₃), and mixed oxides of these oxides, as well as blends thereof. In some embodiments, the metaloxides are microfine grade, while in other embodiments, the metaloxides are pigment grade. In further embodiments, the metaloxides are a mixture of microfine and pigment grades.

In additional embodiments, the inorganic pigments are coated (i.e., they are treated on the surface). In some particularly preferred embodiments, the surface is coated with a thin, hydrophobic film. In some other particularly preferred embodiments, the surface is coated with a thin, hydrophilic film. In yet additional embodiments, the present invention provides compositions comprising various make ups and make up constituents. For example, in some embodiments, the present invention provides various dyes and/or pigments. In some embodiments, useful pigments include, but are not limited to titanium dioxide, mica, iron oxides (e.g. Fe₂O₃, Fe₃O₄, FeO(OH), etc.) and/or stannous oxide. The present invention further provides colorants, including but not limited to carmine, blue, chromooxide, ultramarine and/or purple manganese. The colorants and pigments of some most preferred embodiments are known to those in the art and provided previously (See e.g., Colour Index Nummern (CIN), Rowe Colour Index, 3^(rd) ed., Society of Dyers and Colourists, Bradford, England [1971]).

In additional embodiments, pearlescent pigments based on mica/metaloxide find use, as described above. However, it is not intended that the present invention be limited to these particular pigments, as additional pearlescent pigments find use in various embodiments of the present invention.

The following formulations provide additional examples of the use of the present invention.

RAW MATERIAL Formulation (Amounts) (INCI Designation 1 2 3 4 5 Sodium Carbomer 0.2 Acrylates/C₁₀-C₃₀ Alkyl Acrylate 0.3 0.2 0.6 Crosspolymer Hydroxypropyl Cellulose 1.0 1.50 Xanthan Gummi 0.6 0.2 1.0 1.0 Compound 0.5 0.1 0.01 0.01 1.0 Dioctyl Butamidotriazon 2.0 2.0 1.0 Ethylhexyl Triazon 4.0 4.0 5.0 Aniso Triazin 1.0 0.5 2.0 2.5 Bisoctyltriazol 6.0 Drometrizole Trisiloxane PhenylbenzmidazSulfonicacid 2.0 1.0 Bisimidazylate 1.0 Terephthalylidene Dicamphor 0.2 Sulfonic Acid Ethylhexyl Methoxycinnamat 7.5 10.0 5.0 Octocrylen 5.0 Dimethicone- 4.0 diethylbenzalmalonate Ethylhexyl Salicylate Homosalate Butyl Methoxydibenzoylmethan 1.0 1.0 4.0 Titan dioxide 1.0 4.0 Zinc oxide 4.0 Caprylic/Capric Triglycerid 2.0 Hydrogenated Coco- 3.0 Glyceride C12-15 Alkyl Benzoat 2.0 2.5 3.0 Dicaprylyl Ether 4.0 Butylenglycol Dicaprylat/ 4.0 2.0 6.0 Dicaprat Dicaprylyl Carbonat 2.0 Cetyl Dimethicon 2.0 0.5 1.0 Shea Butter 2.0 PVP Hexadecen Copolymer 0.5 0.05 0.5 Glycerin 3.0 7.5 7.5 2.5 Tocopherol 0.5 0.75 0.2 Trisodium EDTA 1.0 0.5 0.5 1.0 1.5 Natriumcitrat 0.2 Zitronensäure 0.1 0.1 0.1 DMDM Hydantoin 0.6 0.2 Methylparaben 0.5 0.3 0.15 Phenoxyethanol 0.5 0.4 0.4 1.0 0.60 Ethanol 3.0 2.0 3.0 1.0 Perfume 0.2 0.2 0.2 Water QS (100) QS (100) QS (100) QS (100) QS (100)

RAW MATERIAL Formulations (Amounts) (INCI Designation) 1 2 3 4 5 Sodium Carbomer 0.5 1.5 Acrylates/C₁₀-C₃₀ Alkyl 0.4 0.1 0.75 Acrylate Crosspolymer Hydroxypropyl Cellulose 0.5 0.25 Xanthan Gummi 0.2 0.4 Compound 0.5 0.1 0.01 0.01 1.0 Dioctyl Butamidotriazon 1.0 2.0 Ethylhexyl Triazon 2.0 2.0 Aniso Triazin 1.0 0.2 3.0 1.0 Bisoctyltriazol 8.0 Drometrizole Trisiloxane 4.0 Phenylbenzmidazole 1.5 Sulfonicacid Bisimidazylate 1.5 Terephthalylidene Dicamphor 0.5 Sulfonic Acid Ethylhexyl Methoxycinnamat 7.5 5.0 10.0 Octocrylen 10.0 5.0 5.0 Dimethicone- 2.5 diethylbenzalmalonate Ethylhexyl Salicylate 3.5 5.0 Homosalate 4.0 Butyl 0.5 Methoxydibenzoylmethan Titandioxide 1.5 2.0 1.0 2.5 Zincoxide 1.0 0.5 Caprylic/Capric Triglycerid Hydrogenierte Coco-Glyceride C12-15 Alkyl Benzoat 5.0 Dicaprylyl Ether 7.5 Butylenglycol Dicaprylat/Dicaprat Dicaprylyl Carbonat 7.5 Cetyl Dimethicon Shea Butter 3.0 PVP Hexadecen Copolymer 0.5 0.75 1.0 Glycerin 5.0 10.0 Tocopherol 0.3 1.5 1.0 Trisodium EDTA 0.5 0.1 0.5 Natriumcitrat 0.3 Zitronensäure 0.15 DMDM Hydantoin 0.3 0.15 Methylparaben 0.4 Phenoxyethanol 1.0 Ethanol 7.5 5.0 7.0 Perfume 0.25 0.2 Water QS (100) QS (100) QS (100) QS (100) QS (100)

RAW MATERIAL Formulations (Amounts) (INCI Designation) 1 2 3 4 5 6 Sodium Carbomer 0.5 1.5 1.0 0.5 Acrylates/C₁₀-C₃₀ Alkyl 1.0 0.75 1.0 Acrylate Crosspolymer Hydroxypropyl Cellulose 0.4 1.0 1.0 Xanthan Gummi 0.6 0.2 1.0 1.0 BBI 4.0 0.5 3.0 2.0 4.0 1.5 Dioctyl Butamidotriazon 2.0 2.0 2.0 1.0 Ethylhexyl Triazon 4.0 5.0 4.0 Aniso Triazin 1.0 1.0 2.5 1.0 Bisoctyltriazol 4.0 Drometrizole Trisiloxane 3.0 Phenylbenzmidazole 2.0 1.0 Sulfonicacid Bisimidazylate 1.5 3.5 Terephthalylidene 0.2 1.0 Dicamphor Sulfonic Acid Ethylhexyl 10.0 5.0 Methoxycinnamat Octocrylen 10.0 5.0 Dimethicone- 4.0 diethylbenzalmalonate Ethylhexyl 5.0 Salicylate Homosalate 5.0 Butyl Methoxy- 1.0 1.0 4.0 0.5 dibenzoylmethan Titandioxide 1.0 4.0 1.5 Zincoxide 4.0 Caprylic/Capric 2.0 Triglycerid Paraffinöl 1.0 C₁₂-C₁₅ Alkyl 2.0 2.5 3.0 Benzoat Dicaprylyl Ether 4.0 Isohexadecen 4.0 2.0 6.0 Dicaprylyl 2.0 Carbonat Dibutyl Adipat 2.0 0.5 1.0 Cylomethicon 3.0 Jojobaöl 2.0 PVP Hexadecen 0.5 0.05 0.5 0.5 Copolymer Butylen Glycol 3.0 7.5 7.5 2.5 5.0 Ascorbyl-Palmitate 0.5 0.75 0.2 0.3 Octoxyglycerin 1.0 0.5 1.0 Glycin Soja 2.0 1.5 Trisodium EDTA 1.0 0.5 0.5 1.5 0.5 Caustic acid 1.0 0.2 0.25 Iodopropyl 0.6 0.2 Butylcarbamat Phenoxyethanol 0.4 1.0 Ethanol 5.0 2.0 7.0 Perfume 0.2 0.2 0.2 Water QS (100) QS (100) QS (100) QS (100) QS (100) QS (100) The following formula provides an example of an after-shave product comprising the BBI-AV of the present invention.

AFTER SHAVE LOTION % Ingredient INCI A 10.0 Luvitol EHO Cetearyl Ethylhexanoate 5.0 Vitamin E Acetate Tocopheryl Acetate 1.0 Bisabolol rac. Bisabolol 0.1 Perfume 0.3 Carbopol Ultrez 21 Acrylates/C10-30 Alkyl Acrylate Crosspolymer B 15.0 Ethanol Alcohol 1.0 D-Panthenol USP Panthenol 3.0 Glyerin 87% Glycerin 0.1 Triethanolamine Care Triethanolamine SA Compound QS Water dem. Aqua dem. Production: Weigh out the components of Phase A and mix them. Dissolve Phase B, stir it into Phase A and homogenize well.

Measure Values:

Viscosity: 18 500 mPa s Brookfield RVD II+

pH value: 5.8

The following formula provides an example of an after-shave product comprising the BBI-AV of the present invention.

PRE SHAVE % Ingredient INCI A 80 Ethanol Alcohol 3.0 Vitamin E Acetate Tocopheryl Acetate 1.0 Bisabolol rac. Bisabolol 0.2 Perfume 0.1 Menthol Menthol 4.0 Luvitol EHO Cetearyl Ethylhexanoate 2.0 Eutanol G Octyldodecanol 2.0 Miglyol 812 Caprylic/Capric Triglyceride 2.0 D-Panthenol USP Panthenol 2.0 Whitch Hazel Distillate Hamamelis Virginiana (Whitch Hazel) Distillate 2.0 Jojoba Oil Simmondsia Chinensis (Jojoba) Seed Oil SA Compound Production: Weigh out the components of Phase A and dissolve them clearly.

The after-shave and pre-shave formula provided above contain sufficient BBI-AV (Compound) to provide the desired effect(s). In some embodiments, the concentration of BBI-AV is in the range of about 1,000 ppm to about 10,000 ppm. In the following formulations, typical concentrations of BBI-AV used range from about 100 ppm to about 1,000 ppm or from about 1,000 ppm to about 10,000 ppm. However, it is not intended that the present invention be limited to this specific concentration range, as other concentrations find use in other embodiments of the present invention.

The following formula provides an example of an after-sun product comprising the BBI-AV of the present invention.

AFTER SUN LOTION % Ingredient INCI A 0.4 Carbopol 1342 Acrylates/C10-30 Alkyl Acrylate Crosspolymer 15.0 Luvitol EHO Cetearyl Ethylhexanoate 0.2 Bisabolol rac. Bisabolol 1.0 Vitamin E Acetate Tocopheryl Acetate q.s. Perfume B 1.0 D-Panthenol USP Panthenol 15.0 Ethanol 96% Alcohol 3.0 Glycerin 87% Glycerin SA Compound 64.2 Water dem. Aqua dem. C 0.2 Triethanolamine Care Triethanolamine Production: Mix the components of Phase A. Dissolve Phase B and stir it into Phase A whilst homogenizing. Neutralize with Phase C and homogenize again.

Measure Values:

Viscosity: 7 500 mPa s Haake Viscotester VT-02

pH value: 6.0

The following formula provides an example of a facial cleanser product comprising the BBI-AV of the present invention.

FACIAL CLEANSER % Ingredient INCI A 10.0  Luvitol EHO Cetearyl Ethylhexanoate 10.0  Miglyol 812 Caprylic/Capric Triglyceride 1.5 Dow Corning 345 Fluid Cyclopentasiloxane, Cyclohexasilosane 2.0 Cremophor CO 40 PEG-40 Hydrogenated Castor Oil B 3.5 Luvigel EM Caprylic/Capric Triglyceride, Sodium Acrylates Copolymer C 1.0 Vitamin E Acetate Tocopheryl Acetate 0.2 Bisabolol rac. Bisabolol QS Preservative QS Perfume D 3.0 Luviquat Care Polyquaternium-44 0.5 Luviquat Mono LS Cocotrimonium Methosulfate 0.5 Cremophor A 25 Ceteareth-25 0.2 D-Panthenol 50 P Panthenol, Propylene Glycol 4.0 1,2 Propylene Glycol Propylene Glycol Care 0.1 Edeta BD Disodium EDTA SA Compound QS Water dem. Aqua dem. Production: Dissolve Phase A, then stir in Phase B. Fold in Phase C. Dissolve Phase D, stir it into the combined Phases A+B+C, homogenize and stir again for 15 min.

Measure Values:

Viscosity: 7 200 mPa s Brookfield RVT

pH value: 5.8

The following formula provides an example of a daily care body spray product with SPF 8 comprising the BBI-AV of the present invention.

DAILY CARE BODY SPRAY - SPF 8 % Ingredient INCI A 3.0 Uvinul MC 80 Ethylhexyl Methoxycinnamate 2.0 Uvinul A Plus ™ Diethylamino Hydroxybenzoyl Hexyl Benzoate 1.0 Luviquat UltraCare Polyquaternium-44 3.0 1,2 Propylenglycol Care Propylene Glycol 2.0 D-Panthenol 50 P Panthenol, Propylene Glycol 1.0 Dow Corning 345 Fluid Cyclopentasiloxane, Cyclohexasiloxane 10.0  Eutanol G Octyldodecanol 0.5 Luviskol K 30 PVP 10.0  Miglyol 812 Caprylic/Capric Triglyceride 3.0 Finsolv TN C12-15 Alkyl Benzoate 3.0 Glycerin 87% Glycerin 1.0 Vitamin E Acetate Tocopheryl Acetate 0.3 Bisabolol rac. Bisabolol Compound QS Ethanol Alcohol Production: Weigh out the components of Phase A and dissolve them clearly.

Measure Values:

SPF: 8 Colipa Task Force “Sun Protection Measurement”

The following formula provides an example of a daily care sun care lotion product with SPF 27 comprising the BBI-AV of the present invention.

SUN CARE LOTION - SPF 27 % Ingredient INCI A 4.5 Uvinul MC 80 Ethylhexyl Methoxycinnamate 2.0 Uvinul A Plus ™ Diethylamino Hydroxybenzoyl Hexyl Benzoate 3.0 Uvinul N 539 T Octocrylene 2.5 Cosmacol EMI Di-C12-13 Alkyl Malate 0.5 Vitamin E Acetate Tocopheryl Acetate 4.0 Tego Care 450 Polyglyceryl-3 Methyl Glucose Distearate B 3.5 Cetiol SN Deo Cetearyl Isononanoate 1.0 Ganex V-220 VP/Eicosene Copolymer 5.0 Isohexadecane Isohexadecane 2.5 Cosmacol EMI Di-C12-13 Alkyl Malate 3.0 Uvinul TiO2 Titanium Dioxide, Trimethoxycaprylylsilane C 5.0 Glycerin 87% Glycerin 1.0 Lanette E Sodium Cetearyl Sulfate 0.5 Keltrol Xanthan Gum 60.7  Water dem. Aqua dem. D SA Compound 1.0 Phenonip Phenoxyethanol, Methylparaben, Ethylparaben, 0.3 Bisabolol rac. Bisabolol Production: Heat Phases A and B separately to about 80° C. Stir Phase B into Phase A whilst homogenizing. Heat Phase C to about 80° C. and stir it into the combined Phases A+B whilst homogenizing. Cool to about 40° C. add Phase D and homogenize again.

Measure Values:

Viscosity: 3 200 mPa s Brookfield RVD II+

pH value: 6.0

SPF: 27 Colipa Task Force “Sun Protection Measurement”

SUN CARE LOTION - SPF 24 % Ingredient INCI A 2.0 Cremophor A 6 Ceteareth-6, Stearyl Alcohol 2.0 Cremophor A 25 Ceteareth-25 3.0 Syncrowax HRC Tribehenin 2.0 Lanette O Cetearyl Alcohol 2.0 Luvitol EHO Cetearyl Ethylhexanoate 5.0 Uvinul MC 80 Ethylhexyl Methoxycinnamate 1.0 Uvinul T 150 Ethylhexyl Triazone 1.0 Ganex V-220 VP/Eicosene Copolymer 7.0 Isopropyl Myristate Isopropyl Myristate B 5.0 Z-Cote HP-1 Zinc Oxide, Triethoxycaprylylsilane C 0.2 Keltrol Hydroxyethyl Acrylate/Sodium Acryloyldimethyl Taurate Copolymer, Squalane, Polysorbate 60 0.2 Edeta BD Disodium EDTA 5.0 1,2 Propylene Glycol Care Propylene Glycol 0.5 D-Panthenol USP Panthenol 61.9  Water dem. Aqua dem. D SA Compound 0.5 Euxyl K 300 Phenoxyethanol, Methylparaben, Butylparaben, Ethylparaben, Propylparaben, Isobutylparaben 1.0 Vitamin E Acetate Tocopheryl Acetate 0.2 Bisabolol rac. Bisabolol Production: Heat Phase A to 80° C., add Phase B and homogenize for 3 min. Heat Phase C to about 80° C., and stir it into the combined Phases A+B whilst homogenizing. Cool to about 40° C., add Phase D, and homogenize.

Measure Values:

Viscosity: 5 000 mPa s Brookfield RVD II+

pH value: 7.5

SPF: 24 Colipa Task Force “Sun Protection Measurement”

The following formula provides an example of a sun screen emulsion product with SPF 28 comprising the BBI-AV of the present invention.

SUN SCREEN EMULSION - SPF 28 % Ingredient INCI A 3.5 Cremophor A 6 Ceteareth-6, Stearyl Alcohol 1.5 Cremophor A 25 Ceteareth-25 7.5 Uvinul MC 80 Ethylhexyl Methoxycinnamate 2.0 Uvinul A Plus ™ Diethylamino Hydroxybenzoyl Hexyl Benzoate 2.0 Dow Corning 345 Fluid Cyclopentasiloxane, Cyclohexasiloxane 0.5 Bees Wax 3044 PH Bees Wax 3.0 Lanette O Cetearyl Alcohol 10.0  Miglyol 812 Caprylic/Capric Triglyceride B 5.0 T-Lite SF-S Titanium Dioxide, Silica, Methicone, Alumina C 3.0 Glycerin 87% Glycerin 0.2 Edeta BD Disodium EDTA 0.3 Keltrol T Xanthan Gum 1.0 Plantacare 2000 Decyl Glucoside 2.0 D-Panthenol 50 P Panthenol, Propylene Glycol 57.3  Water dem. Aqua dem. D SA Compound 1.0 Vitamin E Acetate Tocopheryl Acetate 0.2 Bisabolol rac. Bisabolol QS Perfume QS Preservative Production: Heat Phase A to 80° C., add Phase B and homogenize for 3 min. Heat Phase C to about 80° C., and stir it into the combined Phases A+B whilst homogenizing. Cool to about 40° C., add Phase D and homogenize.

Measure Values:

Viscosity: 7 500 mPa s Brookfield RVD II+

pH value: 6.6

SPF: 28 Colipa Task Force “Sun Protection Measurement”

The following formula provides an example of a foot balm product comprising the BBI-AV of the present invention.

FOOT BALM % Ingredient INCI A 2.0 Cremophor A 6 Ceteareth-6, Stearyl Alcohol 2.0 Cremophor A 25 Ceteareth-25 5.0 Luvitol EHO Cetearyl Ethylhexanoate 4.0 Lanette 16 Cetyl Alcohol 4.0 Cutina Gms Glyceryl Stearate 5.0 Paraffin Oil Mineral Oil 0.2 Menthol Menthol 0.5 Camphor Camphor B 70.3  Water dem. Aqua dem. QS Preservative C SA Compound 1.0 Bisabolol rac. Bisabolol 1.0 Vitamin E Acetate Tocopheryl Acetate D 5.0 Witch Hazel Extract Witch Hazel Extract Production: Heat Phases A and B to about 80° C. separately. Stir Phase B into Phase A whilst homogenizing. Cool to about 40° C., add Phases C and D and homogenize again. Cool to room temperature.

Measure Values:

Viscosity: 20 500 mPa s Brookfield RVD II+

pH value: 6.0

The following formula provides an example of a refreshing foot gel product comprising the BBI-AV of the present invention.

REFRESHING FOOT GEL % Ingredient INCI A 0.6 Carbopol Ultrez 21 Acrylates/C10-30 Alkyl Acrylate Crosspolymer 45.9  Water dem. Aqua dem. B 1.0 Bisabolol rac. Bisabolol 0.5 Farnesol Farnesol q.s. Perfume 4.5 Cremophor CO 40 PEG-40 Hydrogenated Castor Oil 1.0 Neutrol TE Tetrahydroxypropyl Ethylenediamine 1.5 Menthol Menthol SA Compound 45.0  Ethanol 96% Alcohol QS FD&C Blue No. 1 C.I. 42 090, FD&C Blue No. 1 Production: Phase A: Intersperse the Carbopol and let it settle on the bottom of the beaker. Dissolve Phase B and stir it into Phase A.

Measure Values:

Viscosity: 14 500 mPa s Brookfield RVD II+

pH value: 7.5

The following formula provides an example of a skin conditioning gel product comprising the BBI-AV of the present invention.

Skin Conditioning Gel % Ingredient INCI A 3.6 Cremophor CO 40 PEG-40 Hydrogenated Castor Oil 15.0  Ethanol Alcohol 0.1 Bisabolol rac. Bisabolol 0.5 Vitamin E Acetate Tocopheryl Acetate QS Perfume B 3.0 D-Panthenol USP Panthenol 0.6 Carbopol 940 Carbomer SA Compound 76.4  Water dem. Aqua dem. C 0.8 Triethanolamine Care Triethanolamine Production: Dissolve Phase A clearly. Allow Phase B to swell and neutralize with Phase C. Stir Phase A into the neutralized Phase B and homogenize.

Measure Values:

Viscosity: 57 600 mPa s Brookfield RVD II+

pH value: 7.7

The following formula provides an example of a W/O emulsion comprising the BBI-AV of the present invention.

W/O EMULSION % Ingredient INCI A 6.0 Cremophor WO 7 PEG-7 Hydrogenated Castor Oil 8.0 Luvitol EHO Cetearyl Ethylhexanoate 5.0 Isopropyl Myristate Isopropyl Myristate 15.0  Paraffin Oil Mineral Oil 0.3 Magnesium Stearate Magnesium Stearate 0.3 Aluminum Stearate Aluminum Stearate 2.0 Elfacos ST9 PEG-45/Dodecyl Glycol Copolymer B 5.0 Glycerin 87% Glycerin 0.7 Magnesium Magnesium Sulfate Sulfate-7-hydrate 56.6  Water dem. Aqua dem. C SA Compound 0.5 Vitamin E Acetate Tocopheryl Acetate 0.6 Bisabolol rac. Bisabolol Production: Heat Phases A and B separately to about 85° C. Stir Phase B into Phase

A and homogenize. Cool to about 400C whilst stirring, add Phase C and homogenize again. Cool to room temperature.

Measure Values:

Viscosity: 37 500 mPa s Brookfield RVD II+

The following formula provides an example of a O/W emulsion product comprising the BBI-AV of the present invention.

O/W EMULSION % Ingredient INCI A 1.7 Cremophor A 6 Ceteareth-6, Stearyl Alcohol 0.7 Cremophor A 25 Ceteareth-25 2.0 Uvinul A Plus ™ Diethylamino Hydroxybenzoyl Hexyl Benzoate 2.0 Abil B 8843 PEG-14 Dimethicone 3.6 Lanette O Cetearyl Alcohol 6.0 Uvinul MC 80 Ethylhexyl Methoxycinnamate 2.0 Cetiol B Dibutyl Adipate B 5.0 Glycerin 87% Glycerin 0.2 Edeta BD Disodium EDTA 1.0 D-Panthenol 75 W Panthenol q.s. Preservative 68.8  Water dem. Aqua dem. C 4.0 Luvigel EM Caprylic/Capric Triglyceride, Sodium Acrylates Copolymer D 0.2 Sodium Ascorbyl Sodium Ascorbyl Phosphate Phosphate 1.0 Vitamin E Acetate Tocopheryl Acetate 0.2 Bisabolol rac. Bisabolol E q.s. Sodium Hydroxide Sodium Hydroxide 10% aq. w/w F 1.0 RetiSTAR Caprylic/Capric Triglyceride, Sodium Ascorbate, Tocopherol, Retinol SA Compound Production: Heat Phase A and B separately to about 80° C. Stir Phase B into Phase A and homogenize. Stir Phase C into the combined Phases A+B and homogenize. Cool to about 40° C., add Phase D, then adjust the pH value with Phase E to 6.5. Add Phase F and homogenize. Cool to room temperature.

Measure Values:

Viscosity: 37 500 mPa s Brookfield RVD II+

pH value: 6.3

The following formula provides an example of a protective day cream product comprising the BBI-AV of the present invention.

PROTECTIVE DAY CREAM % Ingredient INCI A 1.7 Cremophor A 6 Ceteareth-6, Stearyl Alcohol 0.7 Cremophor A 25 Ceteareth-25 2.0 Uvinul A Plus ™ Diethylamino Hydroxybenzoyl Hexyl Benzoate 2.0 Abil B 8843 PEG-14 Dimethicone 3.6 Lanette O Cetearyl Alcohol 6.0 Uvinul MC 80 Ethylhexyl Methoxycinnamate 2.0 Cetiol B Dibutyl Adipate B 5.0 Glycerin 87% Glycerin 0.2 Edeta BD Disodium EDTA 1.0 D-Panthenol 75 W Panthenol QS Preservative 69.6  Water dem. Aqua dem. C 4.0 Luvigel EM Caprylic/Capric Triglyceride, Sodium Acrylates Copolymer D 1.0 Sodium Ascorbyl Sodium Ascorbyl Phosphate Phosphate 1.0 Vitamin E Acetate Tocopheryl Acetate SA Compound 0.2 Bisabolol rac. Bisabolol E QS Sodium Hydroxide Sodium Hydroxide 10% aq. w/w Production: Heat Phase A and B separately to about 80° C. Stir Phase B into Phase A and homogenize. Stir Phase C into the combined Phases A+B and homogenize. Cool to about 40° C., add Phase D, then adjust the pH value with Phase E to 6.5 and homogenize. Cool to room temperature.

Measure Values:

Viscosity: 24 000 mPa s Brookfield RVD II+

pH value: 6.4

The following formulae provide examples of hair care products comprising the BBI-AV of the present invention.

HAIR CONDITIONERS % Ingredients (INCI) A 10.0  PVP/VA Copolymer 0.2 Hydroxyethyl Cetyldimonium Phosphate 0.2 Ceteareth-25 0.5 Dimethicone Copolyol QS Perfume 10.0  Alcohol SA Compound 68.1  Aqua dem. B 10.0  Propane/Butane A 10.0  PVP/VA Copolymer 0.2 Hydroxyethyl Cetyldimonium Phosphate 0.2 Ceteareth-25 0.5 Dimethicone Copolyol QS Perfume 10.0  Alcohol SA Compound 64.1  Aqua dem. B 10.0  Propane/Butane Production: Add all compounds to Phase A and stir to homogenize. Fill into appropriate container and charge with Phase B.

FOAM CONDITIONERS % Ingredients (INCI) A 1.0 Polyquaternium-4 0.5 Hydroxyethyl Cetyldimonium Phosphate Compound QS Perfume QS Preservative 91.5  Aqua dem. B 6.0 Propane/Butane A 1.0 Polyquaternium-4 0.5 Hydroxyethyl Cetyldimonium Phosphate Compound QS Perfume QS Preservative 87.5  Aqua dem. B 6.0 Propane/Butane Production: Add all compounds to Phase A and stir to homogenize. Fill into appropriate container and charge with Phase B.

FOAM CONDITIONERS % Ingredients (INCI) A 1.0 Polyquaternium-11 0.5 Hydroxyethyl Cetyldimonium Phosphate Compound QS Perfume QS Preservative 91.5  Aqua dem. B 6.0 Propane/Butane A 1.0 Polyquaternium-11 0.5 Hydroxyethyl Cetyldimonium Phosphate SA Compound QS Perfume QS Preservative 87.5  Aqua dem. B 6.0 Propane/Butane Production: Add all compounds to Phase A and stir to homogenize. Fill into appropriate container and charge with Phase B.

STYLING FOAMS % Ingredients (INCI) A 0.5 Laureth-4 QS Perfume B 77.3  Aqua dem. 10.0  Polyquaternium-28 Compound 0.5 Dimethicone Copolyol 0.2 Ceteareth-25 0.2 Panthenol 0.1 PEG-25 PABA 0.2 Hydroxyethylcellulose C 10.0  HFC 152 A A 0.5 Laureth-4 QS Perfume B 73.3  Aqua dem. 10.0  Polyquaternium-28 SA Compound 0.5 Dimethicone Copolyol 0.2 Ceteareth-25 0.2 Panthenol 0.1 PEG-25 PABA 0.2 Hydroxyethylcellulose C 10.0  HFC 152 A Production: Weight out the compounds of Phase A and mix them. Dissolve Phase B, stir into Phase A and homogenize. Fill into appropriate container and charge with Phase C.

% Ingredients (INCI) A 2.0 Cocotrimonium Methosulfate QS Perfume B 78.5  Aqua dem. 6.7 Acrylates Copolymer 0.6 AMP SA Compound 0.5 Dimethicone Copolyol 0.2 Ceteareth-25 0.2 Panthenol 0.1 PEG-25 PABA 0.2 Hydroxyethylcellulose C 10.0  HFC 152 A A 2.0 Cocotrimonium Methosulfate QS Perfume B 74.5  Aqua dem. 6.7 Acrylates Copolymer 0.6 AMP SA Compound 0.5 Dimethicone Copolyol 0.2 Ceteareth-25 0.2 Panthenol 0.1 PEG-25 PABA 0.2 Hydroxyethylcellulose C 10.0  HFC 152 A Production: Weight out the compounds of Phase A and mix them. Dissolve Phase B, stir into Phase A and homogenize. Fill into appropriate container and charge with Phase C.

STYLING FOAM % Ingredients (INCI) A  2.0 Cocotrimonium Methosulfate QS Perfume B  7.70 Polyquaternium-44 SA Compound QS Preservative 79.3 Aqua dem. C 10.0 Propane/Butane Production: Weight out the compounds of Phase A and mix them. Dissolve Phase B, stir into Phase A and homogenize. Adjust pH to 6-7. Fill into appropriate container and charge with Phase C.

STYLING FOAM % Ingredients (INCI) A 2.00 Cocotrimonium Methosulfate QS Perfume B 72.32  Aqua dem. 2.00 VP/Acrylates/Lauryl Methacrylate Copolymer 0.53 AMP SA Compound 0.20 Ceteareth-25 0.50 Panthenol 0.05 Benzophenone-4 0.20 Amodimethicone, Cetrimonium Chloride, Trideceth-12 15.00  Alcohol C 0.20 Hydroxyethylcellulose D 6.00 Propane/Butane A 2.00 Cocotrimonium Methosulfate QS Perfume B 68.32  Aqua dem. 2.00 VP/Acrylates/Lauryl Methacrylate Copolymer 0.53 AMP SA Compound 0.20 Ceteareth-25 0.50 Panthenol 0.05 Benzophenone-4 0.20 Amodimethicone, Cetrimonium Chloride, Trideceth-12 15.00  Alcohol C 0.20 Hydroxyethylcellulose D 6.00 Propane/Butane Production: Weight out the compounds of Phase A and mix them. Dissolve Phase B, stir into Phase A and homogenize. Add Phase C and homogenize again. Adjust pH to 6-7. Fill into appropriate container and charge with Phase D.

STYLING FOAMS % Ingredients (INCI) A 2.00 Cetrimonium Chloride QS Perfume B 67.85  Aqua dem. 7.00 Polyquaternium-46 SA Compound 0.20 Ceteareth-25 0.50 Panthenol 0.05 Benzophenone-4 0.20 Amodimethicone, Cetrimonium Chloride, Trideceth-12 15.00  Alcohol C 0.20 Hydroxyethylcellulose D 6.00 Propane/Butane A 2.00 Cetrimonium Chloride QS Perfume B 63.85  Aqua dem. 7.00 Polyquaternium-46 SA Compound 0.20 Ceteareth-25 0.50 Panthenol 0.05 Benzophenone-4 0.20 Amodimethicone, Cetrimonium Chloride, Trideceth-12 15.00  Alcohol C 0.20 Hydroxyethylcellulose D 6.00 Propane/Butane Production: Weight out the compounds of Phase A and mix them. Dissolve Phase B, stir into Phase A and homogenize. Add Phase C and homogenize again. Adjust pH to 6-7. Fill into appropriate container and charge with Phase D.

STYLING FOAMS % Ingredients (INCI) A QS PEG-40 Hydrogenated Castor Oil QS Perfume 85.5  Aqua dem. B 7.0 Sodium Polystyrene Sulfonate SA Compound 0.5 Cetrimonium Bromide QS Preservative C 6.0 Propane/Butane A QS PEG-40 Hydrogenated Castor Oil QS Perfume 81.5  Aqua dem. B 7.0 Sodium Polystyrene Sulfonate SA Compound 0.5 Cetrimonium Bromide QS Preservative C 6.0 Propane/Butane Production: Weight out the compounds of Phase A and mix them. Dissolve Phase B, stir into Phase A and homogenize. Adjust pH to 6-7. Fill into appropriate container and charge with Phase C.

STYLING FOAMS % Ingredients (INCI) A QS PEG-40 Hydrogenated Castor Oil QS Perfume 92.0  Aqua dem. B 0.5 Polyquaternium-10 1.0 Compound 0.5 Cetrimonium Bromide QS Preservative C 6.0 Propane/Butane A QS PEG-40 Hydrogenated Castor Oil QS Perfume 88.0  Aqua dem. B 0.5 Polyquaternium-10 5.0 Compound 0.5 Cetrimonium Bromide QS Preservative C 6.0 Propane/Butane Production: Weight out the compounds of Phase A and mix them. Dissolve Phase B, stir into Phase A and homogenize. Adjust pH to 6-7. Fill into appropriate container and charge with Phase C.

% Ingredients (INCI) A QS PEG-40 Hydrogenated Castor Oil QS Perfume 82.5  Aqua dem. B 10.0  Polyquaternium-16 SA Compound 0.5 Hydroxyethyl Cetyldimonium Phosphate QS Preservative C 6.0 Propane/Butane A QS PEG-40 Hydrogenated Castor Oil QS Perfume 78.5  Aqua dem. B 100    Polyquaternium-16 SA Compound 0.5 Hydroxyethyl Cetyldimonium Phosphate QS Preservative C 6.0 Propane/Butane Production: Weight out the compounds of Phase A and mix them. Dissolve Phase B, stir into Phase A and homogenize. Adjust pH to 6-7. Fill into appropriate container and charge with Phase C.

STYLING FOAMS % Ingredients (INCI) A 2.0 Cocotrimonium Methosulfate QS Perfume B 84.0  Aqua dem. 2.0 Chitosan SA Compound 0.5 Dimethicone Copolyol 0.2 Ceteareth-25 0.2 Panthenol 0.1 PEG-25 PABA C 10.0  HFC 152 A A 2.0 Cocotrimonium Methosulfate QS Perfume B 80.0  Aqua dem. 2.0 Chitosan SA Compound 0.5 Dimethicone Copolyol 0.2 Ceteareth-25 0.2 Panthenol 0.1 PEG-25 PABA C 10.0  HFC 152 A Production: Weight out the compounds of Phase A and mix them. Dissolve Phase B, stir into Phase A and homogenize. Adjust pH to 6-7. Fill into appropriate container and charge with Phase C.

SHAMPOOS % Ingredients (INCI) A 30.0  Sodium Laureth Sulfate 6.0 Sodium Cocoamphoacetate 6.0 Cocamidopropyl Betaine 3.0 Sodium Laureth Sulfate, Glycol Distearate, Cocamide MEA, Laureth-10 SA Compound 7.7 Polyquaternium-44 2.0 Amodimethicone QS Perfume QS Preservative 1.0 Sodium Chloride 43.3  Aqua dem. B QS Citric Acid A 30.0  Sodium Laureth Sulfate 6.0 Sodium Cocoamphoacetate 6.0 Cocamidopropyl Betaine 3.0 Sodium Laureth Sulfate, Glycol Distearate, Cocamide MEA, Laureth-10 Compound 7.7 Polyquaternium-44 2.0 Amodimethicone QS Perfume QS Preservative 1.0 Sodium Chloride 39.3  Aqua dem. B QS Citric Acid Production: Weight out the compounds of Phase A and mix them. Adjust pH to 6-7 with citric acid.

SHOWER GELS % Ingredients (INCI) A 40.0  Sodium Laureth Sulfate 5.0 Decyl Glucoside 5.0 Cocamidopropyl Betaine SA Compound 1.0 Panthenol QS Perfume QS Preservative 2.0 Sodium Chloride 46.0  Aqua dem. B QS Citric Acid A 40.0  Sodium Laureth Sulfate 5.0 Decyl Glucoside 5.0 Cocamidopropyl Betaine SA Compound 1.0 Panthenol QS Perfume QS Preservative 2.0 Sodium Chloride 42.0  Aqua dem. B QS Citric Acid Production: Weight out the compounds of Phase A and mix them. Adjust pH to 6-7 with citric acid.

SHAMPOOS % Ingredients (INCI) A 40.0  Sodium Laureth Sulfate 5.0 Sodium C12-15 Pareth-15 Sulfonate 5.0 Decyl Glucoside QS Perfume 0.1 Phytantriol 44.6  Aqua dem. SA Compound 0.3 Polyquaternium-10 1.0 Panthenol QS Preservative 1.0 Laureth-3 2.0 Sodium Chloride A 40.0  Sodium Laureth Sulfate 5.0 Sodium C12-15 Pareth-15 Sulfonate 5.0 Decyl Glucoside QS Perfume 0.1 Phytantriol 40.6  Aqua dem. SA Compound 0.3 Polyquaternium-10 1.0 Panthenol QS Preservative 1.0 Laureth-3 2.0 Sodium Chloride Production: Weight out the compounds of Phase A and mix them. Adjust pH to 6-7 with citric acid.

SHAMPOOS % Ingredients (INCI) A 15.00  Cocamidopropyl Betaine 10.00  Disodium Cocoamphodiacetate 5.00 Polysorbate 20 5.00 Decyl Glucoside QS Perfume QS Preservative SA Compound 0.15 Guar Hydroxypropyltrimonium Chloride 2.00 Laureth-3 58.00  Aqua dem. QS Citric Acid B 3.00 PEG-150 Distearate A 15.00  Cocamidopropyl Betaine 10.00  Disodium Cocoamphodiacetate 5.00 Polysorbate 20 5.00 Decyl Glucoside QS Perfume QS Preservative Compound 0.15 Guar Hydroxypropyltrimonium Chloride 2.00 Laureth-3 54.00  Aqua dem. QS Citric Acid B 3.00 PEG-150 Distearate Production: Weight out the compounds of Phase A and mix them. Adjust pH to 6-7. Add Phase B and heat to max. 40° C.

BODY LOTIONS % Ingredients (INCI) A 2.0 Ceteareth-25 2.0 Ceteareth-6, Stearyl Alcohol 3.0 Cetearyl Ethylhexanoate 1.0 Dimethicone 4.0 Cetearyl Alcohol 3.0 Glyceryl Stearate SE 5.0 Mineral Oil 4.0 Simmondsia Chinensis (Jojoba) Seed Oil 3.0 Mineral Oil, Lanolin Alcohol B 5.0 Propylene Glycol SA Compound 1.0 Panthenol 0.5 Magnesium Aluminum Silicate QS Preservative 65.5  Aqua dem. C QS Perfume D QS Citric Acid A 2.0 Ceteareth-25 2.0 Ceteareth-6, Stearyl Alcohol 3.0 Cetearyl Ethylhexanoate 1.0 Dimethicone 4.0 Cetearyl Alcohol 3.0 Glyceryl Stearate SE 5.0 Mineral Oil 4.0 Simmondsia Chinensis (Jojoba) Seed Oil 3.0 Mineral Oil, Lanolin Alcohol B 5.0 Propylene Glycol SA Compound 1.0 Panthenol 0.5 Magnesium Aluminum Silicate QS Preservative 61.5  Aqua dem. C QS Perfume D QS Citric Acid Production: Heat Phases A and B separately to approx. 40° C. Add Phase B to Phase A and homogenize by stirring. Add Phase C to the combined Phase A and B and homogenize again. Adjust pH with Phase D to 6-7. Homogenize by stirring and cool to room temperature.

BODY LOTIONS % Ingredients (INCI) A 6.0 PEG-7 Hydrogenated Castor Oil 10.0  Cetearyl Ethylhexanoate 5.0 Isopropyl Myristate 7.0 Mineral Oil 0.5 Shea Butter (Butyrospermum Parkii) 0.5 Aluminum Stearate 0.5 Magnesium Stearate 0.2 Bisabolol 0.7 Quaternium-18-Hectorite B 5.0 Dipropylene Glycol 0.7 Magnesium Sulfate QS Preservative 62.9  Aqua dem. C QS Perfume SA Compound A 6.0 PEG-7 Hydrogenated Castor Oil 10.0  Cetearyl Ethylhexanoate 5.0 Isopropyl Myristate 7.0 Mineral Oil 0.5 Shea Butter (Butyrospermum Parkii) 0.5 Aluminum Stearate 0.5 Magnesium Stearate 0.2 Bisabolol 0.7 Quaternium-18-Hectorite B 5.0 Dipropylene Glycol 0.7 Magnesium Sulfate QS Preservative 58.9  Aqua dem. C QS Perfume SA Compound Production: Heat Phases A and B separately to approx. 80° C. Add Phase B to Phase A and homogenize by stirring. Cool to 40° C. and add Phase C. Homogenize again and cool to room temperature.

HAIR CONDITIONERS % Ingredients (INCI) A 10.0  PVP/VA Copolymer 0.2 Hydroxyethyl Cetyldimonium Phosphate 0.2 Ceteareth-25 0.5 Dimethicone Copolyol QS Perfume 10.0  Alcohol SA Compound 68.1  Aqua dem. B 10.0  Propane/Butane A 10.0  PVP/VA Copolymer 0.2 Hydroxyethyl Cetyldimonium Phosphate 0.2 Ceteareth-25 0.5 Dimethicone Copolyol QS Perfume 10.0  Alcohol SA Compound 64.1  Aqua dem. B 10.0  Propane/Butane Production: Add all compounds to Phase A and stir to homogenize. Fill into appropriate container and charge with Phase B.

FOAM CONDITIONERS % Ingredients (INCI) A 1.0 Polyquaternium-4 0.5 Hydroxyethyl Cetyldimonium Phosphate SA Compound QS Perfume QS Preservative 91.5  Aqua dem. B 6.0 Propane/Butane A 1.0 Polyquaternium-4 0.5 Hydroxyethyl Cetyldimonium Phosphate SA Compound QS Perfume QS Preservative 87.5  Aqua dem. B 6.0 Propane/Butane Production: Add all compounds to Phase A and stir to homogenize. Fill into appropriate container and charge with Phase B.

FOAM CONDITIONERS % Ingredients (INCI) A 1.0 Polyquaternium-11 0.5 Hydroxyethyl Cetyldimonium Phosphate Compound QS Perfume QS Preservative 91.5  Aqua dem. B 6.0 Propane/Butane A 1.0 Polyquaternium-11 0.5 Hydroxyethyl Cetyldimonium Phosphate SA Compound QS Perfume QS Preservative 87.5  Aqua dem. B 6.0 Propane/Butane Production: Add all compounds to Phase A and stir to homogenize. Fill into appropriate container and charge with Phase B.

STYLING FOAMS % Ingredients (INCI) A 0.5 Laureth-4 QS Perfume B 77.3  Aqua dem. 10.0  Polyquaternium-28 SA Compound 0.5 Dimethicone Copolyol 0.2 Ceteareth-25 0.2 Panthenol 0.1 PEG-25 PABA 0.2 Hydroxyethylcellulose C 10.0  HFC 152 A A 0.5 Laureth-4 QS Perfume B 73.3  Aqua dem. 10.0  Polyquaternium-28 SA Compound 0.5 Dimethicone Copolyol 0.2 Ceteareth-25 0.2 Panthenol 0.1 PEG-25 PABA 0.2 Hydroxyethylcellulose C 10.0  HFC 152 A Production: Weight out the compounds of Phase A and mix them. Dissolve Phase B, stir into Phase A and homogenize. Fill into appropriate container and charge with Phase C.

STYLING FOAMS % Ingredients (INCI) A 2.0 Cocotrimonium Methosulfate QS Perfume B 78.5  Aqua dem. 6.7 Acrylates Copolymer 0.6 AMP SA Compound 0.5 Dimethicone Copolyol 0.2 Ceteareth-25 0.2 Panthenol 0.1 PEG-25 PABA 0.2 Hydroxyethylcellulose C 10.0  HFC 152 A A 2.0 Cocotrimonium Methosulfate QS Perfume B 74.5  Aqua dem. 6.7 Acrylates Copolymer 0.6 AMP SA Compound 0.5 Dimethicone Copolyol 0.2 Ceteareth-25 0.2 Panthenol 0.1 PEG-25 PABA 0.2 Hydroxyethylcellulose C 10.0  HFC 152 A Production: Weight out the compounds of Phase A and mix them. Dissolve Phase B, stir into Phase A and homogenize. Fill into appropriate container and charge with Phase C.

STYLING FOAMS % Ingredients (INCI) A 2.0 Cocotrimonium Methosulfate QS Perfume B  7.70 Polyquaternium-44 SA Compound QS Preservative 79.3  Aqua dem. C 10.0  Propane/Butane A 2.0 Cocotrimonium Methosulfate QS Perfume B  7.70 Polyquaternium-44 SA Compound QS Preservative 75.3  Aqua dem. C 10.0  Propane/Butane Production: Weight out the compounds of Phase A and mix them. Dissolve Phase B, stir into Phase A and homogenize. Adjust pH to 6-7. Fill into appropriate container and charge with Phase C.

STYLING FOAMS % Ingredients (INCI) A 2.00 Cocotrimonium Methosulfate QS Perfume B 72.32  Aqua dem. 2.00 VP/Acrylates/Lauryl Methacrylate Copolymer 0.53 AMP SA Compound 0.20 Ceteareth-25 0.50 Panthenol 0.05 Benzophenone-4 0.20 Amodimethicone, Cetrimonium Chloride, Trideceth-12 15.00  Alcohol C 0.20 Hydroxyethylcellulose D 6.00 Propane/Butane A 2.00 Cocotrimonium Methosulfate QS Perfume B 68.32  Aqua dem. 2.00 VP/Acrylates/Lauryl Methacrylate Copolymer 0.53 AMP SA Compound 0.20 Ceteareth-25 0.50 Panthenol 0.05 Benzophenone-4 0.20 Amodimethicone, Cetrimonium Chloride, Trideceth-12 15.00  Alcohol C 0.20 Hydroxyethylcellulose D 6.00 Propane/Butane Production: Weight out the compounds of Phase A and mix them. Dissolve Phase B, stir into Phase A and homogenize. Add Phase C and homogenize again. Adjust pH to 6-7. Fill into appropriate container and charge with Phase D.

STYLING FOAMS % Ingredients (INCI) A 2.00 Cetrimonium Chloride QS Perfume B 67.85  Aqua dem. 7.00 Polyquaternium-46 SA Compound 0.20 Ceteareth-25 0.50 Panthenol 0.05 Benzophenone-4 0.20 Amodimethicone, Cetrimonium Chloride, Trideceth-12 15.00  Alcohol C 0.20 Hydroxyethylcellulose D 6.00 Propane/Butane A 2.00 Cetrimonium Chloride QS Perfume B 63.85  Aqua dem. 7.00 Polyquaternium-46 SA Compound 0.20 Ceteareth-25 0.50 Panthenol 0.05 Benzophenone-4 0.20 Amodimethicone, Cetrimonium Chloride, Trideceth-12 15.00  Alcohol C 0.20 Hydroxyethylcellulose D 6.00 Propane/Butane Production: Weight out the compounds of Phase A and mix them. Dissolve Phase B, stir into Phase A and homogenize. Add Phase C and homogenize again. Adjust pH to 6-7. Fill into appropriate container and charge with Phase D.

STYLING FOAMS % Ingredients (INCI) A QS PEG-40 Hydrogenated Castor Oil QS Perfume 85.5  Aqua dem. B 7.0 Sodium Polystyrene Sulfonate SA Compound 0.5 Cetrimonium Bromide QS Preservative C 6.0 Propane/Butane A QS PEG-40 Hydrogenated Castor Oil QS Perfume 81.5  Aqua dem. B 7.0 Sodium Polystyrene Sulfonate SA Compound 0.5 Cetrimonium Bromide QS Preservative C 6.0 Propane/Butane Production: Weight out the compounds of Phase A and mix them. Dissolve Phase B, stir into Phase A and homogenize. Adjust pH to 6-7. Fill into appropriate container and charge with Phase C.

STYLING FOAMS % Ingredients (INCI) A QS PEG-40 Hydrogenated Castor Oil QS Perfume 92.0  Aqua dem. B 0.5 Polyquaternium-10 1.0 Compound 0.5 Cetrimonium Bromide QS Preservative C 6.0 Propane/Butane A QS PEG-40 Hydrogenated Castor Oil QS Perfume 88.0  Aqua dem. B 0.5 Polyquaternium-10 5.0 Compound 0.5 Cetrimonium Bromide QS Preservative C 6.0 Propane/Butane Production: Weigh out the compounds of Phase A and mix them. Dissolve Phase B, stir into Phase A and homogenize. Adjust pH to 6-7. Fill into appropriate container and charge with Phase C.

STYLING FOAMS % Ingredients (INCI) A QS PEG-40 Hydrogenated Castor Oil QS Perfume 82.5  Aqua dem. B 10.0  Polyquaternium-16 SA Compound 0.5 Hydroxyethyl Cetyldimonium Phosphate QS. Preservative C 6.0 Propane/Butane A QS PEG-40 Hydrogenated Castor Oil QS Perfume 78.5  Aqua dem. B 10.0  Polyquaternium-16 SA Compound 0.5 Hydroxyethyl Cetyldimonium Phosphate QS Preservative C 6.0 Propane/Butane Production: Weight out the compounds of Phase A and mix them. Dissolve Phase B, stir into Phase A and homogenize. Adjust pH to 6-7. Fill into appropriate container and charge with Phase C.

STYLING FOAMS % Ingredients (INCI) A 2.0 Cocotrimonium Methosulfate QS Perfume B 84.0  Aqua dem. 2.0 Chitosan SA Compound 0.5 Dimethicone Copolyol 0.2 Ceteareth-25 0.2 Panthenol 0.1 PEG-25 PABA C 10.0  HFC 152 A A 2.0 Cocotrimonium Methosulfate QS Perfume B 80.0  Aqua dem. 2.0 Chitosan SA Compound 0.5 Dimethicone Copolyol 0.2 Ceteareth-25 0.2 Panthenol 0.1 PEG-25 PABA C 10.0  HFC 152 A Production: Weight out the compounds of Phase A and mix them. Dissolve Phase B, stir into Phase A and homogenize. Adjust pH to 6-7. Fill into appropriate container and charge with Phase C.

SHAMPOOS % Ingredients (INCI) A 30.0  Sodium Laureth Sulfate 6.0 Sodium Cocoamphoacetate 6.0 Cocamidopropyl Betaine 3.0 Sodium Laureth Sulfate, Glycol Distearate, Cocamide MEA, Laureth-10 SA Compound 7.7 Polyquaternium-44 2.0 Amodimethicone QS Perfume QS Preservative 1.0 Sodium Chloride 43.3  Aqua dem. B QS Citric Acid A 30.0  Sodium Laureth Sulfate 6.0 Sodium Cocoamphoacetate 6.0 Cocamidopropyl Betaine 3.0 Sodium Laureth Sulfate, Glycol Distearate, Cocamide MEA, Laureth-10 SA Compound 7.7 Polyquaternium-44 2.0 Amodimethicone SA Perfume SA. Preservative 1.0 Sodium Chloride 39.3  Aqua dem. B QS Citric Acid Production: Weight out the compounds of Phase A and mix them. Adjust pH to 6-7 with citric acid.

SHOWER GELS % Ingredients (INCI) A 40.0  Sodium Laureth Sulfate 5.0 Decyl Glucoside 5.0 Cocamidopropyl Betaine SA Compound 1.0 Panthenol QS Perfume QS Preservative 2.0 Sodium Chloride 46.0  Aqua dem. B QS Citric Acid A 40.0  Sodium Laureth Sulfate 5.0 Decyl Glucoside 5.0 Cocamidopropyl Betaine SA Compound 1.0 Panthenol QS Perfume QS Preservative 2.0 Sodium Chloride 42.0  Aqua dem. B QS Citric Acid Production: Weight out the compounds of Phase A and mix them. Adjust pH to 6-7 with citric acid.

SHAMPOOS % Ingredients (INCI) A 40.0  Sodium Laureth Sulfate 5.0 Sodium C12-15 Pareth-15 Sulfonate 5.0 Decyl Glucoside QS Perfume 0.1 Phytantriol 44.6  Aqua dem. SA Compound 0.3 Polyquaternium-10 1.0 Panthenol QS Preservative 1.0 Laureth-3 2.0 Sodium Chloride A 40.0  Sodium Laureth Sulfate 5.0 Sodium C12-15 Pareth-15 Sulfonate 5.0 Decyl Glucoside QS Perfume 0.1 Phytantriol 40.6  Aqua dem. SA Compound 0.3 Polyquaternium-10 1.0 Panthenol QS Preservative 1.0 Laureth-3 2.0 Sodium Chloride Production: Weight out the compounds of Phase A and mix them. Adjust pH to 6-7 with citric acid.

SHAMPOOS % Ingredients (INCI) A 15.00 Cocamidopropyl Betaine 10.00 Disodium Cocoamphodiacetate  5.00 Polysorbate 20  5.00 Decyl Glucoside QS Perfume QS Preservative SA Compound  0.15 Guar Hydroxypropyltrimonium Chloride  2.00 Laureth-3 58.00 Aqua dem. QS Citric Acid B  3.00 PEG-150 Distearate A 15.00 Cocamidopropyl Betaine 10.00 Disodium Cocoamphodiacetate  5.00 Polysorbate 20  5.00 Decyl Glucoside QS Perfume QS Preservative SA Compound  0.15 Guar Hydroxypropyltrimonium Chloride  2.00 Laureth-3 54.00 Aqua dem. QS Citric Acid B  3.00 PEG-150 Distearate Production: Weight out the compounds of Phase A and mix them. Adjust pH to 6-7. Add Phase B and heat to max. 40° C.

BODY LOTIONS % Ingredients (INCI) A 2.0 Ceteareth-25 2.0 Ceteareth-6, Stearyl Alcohol 3.0 Cetearyl Ethylhexanoate 1.0 Dimethicone 4.0 Cetearyl Alcohol 3.0 Glyceryl Stearate SE 5.0 Mineral Oil 4.0 Simmondsia Chinensis (Jojoba) Seed Oil 3.0 Mineral Oil, Lanolin Alcohol B 5.0 Propylene Glycol SA Compound 1.0 Panthenol 0.5 Magnesium Aluminum Silicate QS Preservative 65.5  Aqua dem. C QS Perfume D QS Citric Acid A 2.0 Ceteareth-25 2.0 Ceteareth-6, Stearyl Alcohol 3.0 Cetearyl Ethylhexanoate 1.0 Dimethicone 4.0 Cetearyl Alcohol 3.0 Glyceryl Stearate SE 5.0 Mineral Oil 4.0 Simmondsia Chinensis (Jojoba) Seed Oil 3.0 Mineral Oil, Lanolin Alcohol B 5.0 Propylene Glycol SA Compound 1.0 Panthenol 0.5 Magnesium Aluminum Silicate QS Preservative 61.5  Aqua dem. C QS Perfume D QS Citric Acid Production: Heat Phases A and B separately to approx. 40° C. Add Phase B to Phase A and homogenize by stirring. Add Phase C to the combined Phase A and B and homogenize again. Adjust pH with Phase D to 6-7. Homogenize by stirring and cool to room temperature.

BODY LOTIONS % Ingredients (INCI) A 6.0 PEG-7 Hydrogenated Castor Oil 10.0  Cetearyl Ethylhexanoate 5.0 Isopropyl Myristate 7.0 Mineral Oil 0.5 Shea Butter (Butyrospermum Parkii) 0.5 Aluminum Stearate 0.5 Magnesium Stearate 0.2 Bisabolol 0.7 Quaternium-18-Hectorite B 5.0 Dipropylene Glycol 0.7 Magnesium Sulfate QS Preservative 62.9  Aqua dem. C QS. Perfume SA Compound A 6.0 PEG-7 Hydrogenated Castor Oil 10.0  Cetearyl Ethylhexanoate 5.0 Isopropyl Myristate 7.0 Mineral Oil 0.5 Shea Butter (Butyrospermum Parkii) 0.5 Aluminum Stearate 0.5 Magnesium Stearate 0.2 Bisabolol 0.7 Quaternium-18-Hectorite B 5.0 Dipropylene Glycol 0.7 Magnesium Sulfate QS Preservative 58.9  Aqua dem. C QS Perfume SA Compound Production: Heat Phases A and B separately to approx. 80° C. Add Phase B to Phase A and homogenize by stirring. Cool to 40° C. and add Phase C. Homogenize again and cool to room temperature.

DAILY SKIN CARE O/W % Ingredients (INCI) A 1.7 Ceteareth-6, Stearyl Alcohol 0.7 Ceteareth-25 2.0 Diethylamino Hydroxybenzoyl Hexyl Benzoate 2.0 PEG-14 Dimethicone 3.6 Cetearyl Alcohol 6.0 Ethylhexyl Methoxycinnamate 2.0 Dibutyl Adipate B 5.0 Glycerin 0.2 Disodium EDTA 1.0 Panthenol QS Preservative 67.8  Aqua dem. C 4.0 Caprylic/Capric Triglyceride, Sodium Acrylates Copolymer D 0.2 Sodium Ascorbyl Phosphate 1.0 Tocopheryl Acetate 0.2 Bisabolol 1.0 Caprylic/Capric Triglyceride, Sodium Ascorbate, Tocopherol, Retinol SA Compound E QS Sodium Hydroxide A 1.7 Ceteareth-6, Stearyl Alcohol 0.7 Ceteareth-25 2.0 Diethylamino Hydroxybenzoyl Hexyl Benzoate 2.0 PEG-14 Dimethicone 3.6 Cetearyl Alcohol 6.0 Ethylhexyl Methoxycinnamate 2.0 Dibutyl Adipate B 5.0 Glycerin 0.2 Disodium EDTA 1.0 Panthenol QS Preservative 63.8  Aqua dem. C 4.0 Caprylic/Capric Triglyceride, Sodium Acrylates Copolymer D 0.2 Sodium Ascorbyl Phosphate 1.0 Tocopheryl Acetate 0.2 Bisabolol 1.0 Caprylic/Capric Triglyceride, Sodium Ascorbate, Tocopherol, Retinol SA Compound E QS Sodium Hydroxide Production: Heat Phase A and B separately to approx. 80° C. Add Phase B to Phase A and homogenize by stirring. Add Phase C to the combined Phase A and B and homogenize again. Cool to approx. 40° C. and add Phase D. Adjust pH with Phase E to approx. 6.5. Homogenize by stirring and cool to room temperature.

PROTECTIVE DAY SKIN CREME O/W % Ingredients (INCI) A 1.7 Ceteareth-6, Stearyl Alcohol 0.7 Ceteareth-25 2.0 Diethylamino Hydroxybenzoyl Hexyl Benzoate 2.0 PEG-14 Dimethicone 3.6 Cetearyl Alcohol 6.0 Ethylhexyl Methoxycinnamate 2.0 Dibutyl Adipate B 5.0 Glycerin 0.2 Disodium EDTA 1.0 Panthenol QS Preservative 68.6  Aqua dem. C 4.0 Caprylic/Capric Triglyceride, Sodium Acrylates Copolymer D 1.0 Sodium Ascorbyl Phosphate 1.0 Tocopheryl Acetate 0.2 Bisabolol SA Compound E QS Sodium Hydroxide A 1.7 Ceteareth-6, Stearyl Alcohol 0.7 Ceteareth-25 2.0 Diethylamino Hydroxybenzoyl Hexyl Benzoate 2.0 PEG-14 Dimethicone 3.6 Cetearyl Alcohol 6.0 Ethylhexyl Methoxycinnamate 2.0 Dibutyl Adipate B 5.0 Glycerin 0.2 Disodium EDTA 1.0 Panthenol QS Preservative 64.6  Aqua dem. C 4.0 Caprylic/Capric Triglyceride, Sodium Acrylates Copolymer D 1.0 Sodium Ascorbyl Phosphate 1.0 Tocopheryl Acetate 0.2 Bisabolol Compound E QS Sodium Hydroxide Production: Heat Phase A and B separately to approx. 80° C. Add Phase B to Phase A and homogenize by stirring. Add Phase C to the combined Phase A and B and homogenize again. Cool to approx. 40° C. and add Phase D. Adjust pH with Phase E to approx. 6.5. Homogenize by stirring and cool to room temperature.

FACIAL CLEANSER O/W % Ingredients (INCI) A 10.0  Cetearyl Ethylhexanoate 10.0  Caprylic/Capric Triglyceride 1.5 Cyclopentasiloxane, Cyclohexasilosane 2.0 PEG-40 Hydrogenated Castor Oil B 3.5 Caprylic/Capric Triglyceride, Sodium Acrylates Copolymer C 1.0 Tocopheryl Acetate 0.2 Bisabolol QS Preservative QS Perfume D 3.0 Polyquaternium-44 0.5 Cocotrimonium Methosulfate 0.5 Ceteareth-25 2.0 Panthenol, Propylene Glycol 4.0 Propylene Glycol 0.1 Disodium EDTA SA Compound 60.7  Aqua dem. A 10.0  Cetearyl Ethylhexanoate 10.0  Caprylic/Capric Triglyceride 1.5 Cyclopentasiloxane, Cyclohexasilosane 2.0 PEG-40 Hydrogenated Castor Oil B 3.5 Caprylic/Capric Triglyceride, Sodium Acrylates Copolymer C 1.0 Tocopheryl Acetate 0.2 Bisabolol QS Preservative QS Perfume D 3.0 Polyquaternium-44 0.5 Cocotrimonium Methosulfate 0.5 Ceteareth-25 2.0 Panthenol, Propylene Glycol 4.0 Propylene Glycol 0.1 Disodium EDTA SA Compound 56.7  Aqua dem. Production: Dissolve Phase A and add Phase B to Phase A and homogenize by stirring. Add. Phase C to the combined Phase A and B and homogenize again. Add. Phase D to the combined Phase A, B and C and homogenize again. Dissolve Phase D and add to Phase A, B, and C and homogenize again. Stir for 15 minutes.

DAILY CARE BODY SPRAY % Ingredients (INCI) A 3.0 Ethylhexyl Methoxycinnamate 2.0 Diethylamino Hydroxybenzoyl Hexyl Benzoate 1.0 Polyquaternium-44 3.0 Propylene Glycol 2.0 Panthenol, Propylene Glycol 1.0 Cyclopentasiloxane, Cyclohexasiloxane 10.0  Octyldodecanol 0.5 PVP 10.0  Caprylic/Capric Triglyceride 3.0 C12-15 Alkyl Benzoate 3.0 Glycerin 1.0 Tocopheryl Acetate 0.3 Bisabolol SA Compound 59.2  Alcohol A 3.0 Ethylhexyl Methoxycinnamate 2.0 Diethylamino Hydroxybenzoyl Hexyl Benzoate 1.0 Polyquaternium-44 3.0 Propylene Glycol 2.0 Panthenol, Propylene Glycol 1.0 Cyclopentasiloxane, Cyclohexasiloxane 10.0  Octyldodecanol 0.5 PVP 10.0  Caprylic/Capric Triglyceride 3.0 C12-15 Alkyl Benzoate 3.0 Glycerin 1.0 Tocopheryl Acetate 0.3 Bisabolol SA Compound 55.2  Alcohol Production: Weight all ingredients of Phase A and dissolve completely by stirring.

SKIN CARE GEL % Ingredients (INCI) A 3.6 PEG-40 Hydrogenated Castor Oil 15.0 Alcohol 0.1 Bisabolol 0.5 Tocopheryl Acetate QS Perfume B 3.0 Panthenol 0.6 Carbomer SA Compound 75.4 Aqua dem, C 0.8 Triethanolamine A 3.6 PEG-40 Hydrogenated Castor Oil 15.0 Alcohol 0.1 Bisabolol 0.5 Tocopheryl Acetate QS Perfume B 3.0 Panthenol 0.6 Carbomer SA Compound 71.4 Aqua dem, C 0.8 Triethanolamine Production: Dissolve Phase A. Swell Phase B and neutralize with Phase C. Add Phase A to Phase B and C and homogenize by stirring.

AFTER SHAVE LOTIONS % Ingredients (INCI) A 10.0 Cetearyl Ethylhexanoate 5.0 Tocopheryl Acetate 1.0 Bisabolol 0.1 Perfume 0.3 Acrylates/C10-30 Alkyl Acrylate Crosspolymer B 15.0 Alcohol 1.0 Panthenol 3.0 Glycerin SA Compound 1.0 Triethanolamine 63.5 Aqua dem. A 10.0 Cetearyl Ethylhexanoate 5.0 Tocopheryl Acetate 1.0 Bisabolol 0.1 Perfume 0.3 Acrylates/C10-30 Alkyl Acrylate Crosspolymer B 15.0 Alcohol 1.0 Panthenol 3.0 Glycerin SA Compound 0.1 Triethanolamine 59.5 Aqua dem. Production: Dissolve Phase A. Dissolve Phase B and add to Phase A. Homogenize by stirring.

AFTER SUN LOTIONS % Ingredients (INCI) A 0.4 Acrylates/C10-30 Alkyl Acrylate Crosspolymer 15.0 Cetearyl Ethylhexanoate 0.2 Bisabolol 1.0 Tocopheryl Acetate QS. Perfume B 1.0 Panthenol 15.0 Alcohol 3.0 Glycerin SA Compound 63.2 Aqua dem, C 0.2 Triethanolamine A 0.4 Acrylates/C10-30 Alkyl Acrylate Crosspolymer 15.0 Cetearyl Ethylhexanoate 0.2 Bisabolol 1.0 Tocopheryl Acetate QS Perfume B 1.0 Panthenol 15.0 Alcohol 3.0 Glycerin SA Compound 59.2 Aqua dem, C 0.2 Triethanolamine Production: Dissolve Phase A. Dissolve Phase B and add to Phase A. Homogenize by stirring. Neutralize Phase A and B by adding Phase C and homogenize again.

SUNSCREEN LOTIONS % Ingredients (INCI) A 4.5 Ethylhexyl Methoxycinnamate 2.0 Diethylamino Hydroxybenzoyl Hexyl Benzoate 3.0 Octocrylene 2.5 Di-C12-13 Alkyl Malate 0.5 Tocopheryl Acetate 4.0 Polyglyceryl-3 Methyl Glucose Distearate B 3.5 Cetearyl Isononanoate 1.0 VP/Eicosene Copolymer 5.0 Isohexadecane 2.5 Di-C12-13 Alkyl Malate 3.0 Titanium Dioxide, Trimethoxycaprylylsilane C 5.0 Glycerin 1.0 Sodium Cetearyl Sulfate 0.5 Xanthan Gum 59.7 Aqua dem. D SA Compound 1.0 Phenoxyethanol, Methylparaben, Ethylparaben, Butylparaben, Propylparaben, Isobutylparaben 0.3 Bisabolol A 4.5 Ethylhexyl Methoxycinnamate 2.0 Diethylamino Hydroxybenzoyl Hexyl Benzoate 3.0 Octocrylene 2.5 Di-C12-13 Alkyl Malate 0.5 Tocopheryl Acetate 4.0 Polyglyceryl-3 Methyl Glucose Distearate B 3.5 Cetearyl Isononanoate 1.0 VP/Eicosene Copolymer 5.0 Isohexadecane 2.5 Di-C12-13 Alkyl Malate 3.0 Titanium Dioxide, Trimethoxycaprylylsilane C 5.0 Glycerin 1.0 Sodium Cetearyl Sulfate 0.5 Xanthan Gum 55.7 Aqua dem. D SA Compound 1.0 Phenoxyethanol, Methylparaben, Ethylparaben, Butylparaben, Propylparaben, Isobutylparaben 0.3 Bisabolol Production: Heat Phase A and B separately to approx. 80° C. Add Phase B to Phase A and homogenize by stirring. Heat Phase C to 80° C. and add to the combined Phase A and B and homogenize again. Cool to approx. 40° C. and add Phase D. Homogenize again.

SUNSCREEN LOTIONS O/W % Ingredients (INCI) A 2.0 Ceteareth-6, Stearyl Alcohol 2.0 Ceteareth-25 3.0 Tribehenin 2.0 Cetearyl Alcohol 2.0 Cetearyl Ethylhexanoate 5.0 Ethylhexyl Methoxycinnamate 1.0 Ethylhexyl Triazone 1.0 VP/Eicosene Copolymer 7.0 Isopropyl Myristate B 5.0 Zinc Oxide, Triethoxycaprylylsilane C 0.2 Xanthan Gum 0.5 Hydroxyethyl Acrylate/Sodium Acryloyldimethyl Taurate Copolymer, Squalane, Polysorbate 60 0.2 Disodium EDTA 5.0 Propylene Glycol 0.5 Panthenol 60.9 Aqua dem. D SA Compound 0.5 Phenoxyethanol, Methylparaben, Butylparaben, Ethylparaben, Propylparaben, Isopropylparaben 1.0 Tocopheryl Acetate 0.2 Bisabolol A 2.0 Ceteareth-6, Stearyl Alcohol 2.0 Ceteareth-25 3.0 Tribehenin 2.0 Cetearyl Alcohol 2.0 Cetearyl Ethylhexanoate 5.0 Ethylhexyl Methoxycinnamate 1.0 Ethylhexyl Triazone 1.0 VP/Eicosene Copolymer 7.0 Isopropyl Myristate B 5.0 Zinc Oxide, Triethoxycaprylylsilane C 0.2 Xanthan Gum 0.5 Hydroxyethyl Acrylate/Sodium Acryloyldimethyl Taurate Copolymer, Squalane, Polysorbate 60 0.2 Disodium EDTA 5.0 Propylene Glycol 0.5 Panthenol 56.9 Aqua dem. D SA Compound 0.5 Phenoxyethanol, Methylparaben, Butylparaben, Ethylparaben, Propylparaben, Isopropylparaben 1.0 Tocopheryl Acetate 0.2 Bisabolol Production: Heat Phase A and B separately to approx. 80° C. Add Phase B to Phase A and homogenize by stirring. Heat Phase C to 80° C. and add to the combined Phase A and B and homogenize again. Cool to approx. 40° C. and add Phase D. Homogenize again.

SUNSCREEN LOTIONS O/W % Ingredients (INCI) A 3.5 Ceteareth-6, Stearyl Alcohol 1.5 Ceteareth-25 7.5 Ethylhexyl Methoxycinnamate 2.0 Diethylamino Hydroxybenzoyl Hexyl Benzoate 2.0 Cyclopentasiloxane, Cyclohexasiloxane 0.5 Bees Wax 3.0 Cetearyl Alcohol 10.0 Caprylic/Capric Triglyceride B 5.0 Titanium Dioxide, Silica, Methicone, Alumina C 3.0 Glycerin 0.2 Disodium EDTA 0.3 Xanthan Gum 1.0 Decyl Glucoside 2.0 Panthenol, Propylene Glycol 56.3 Aqua dem. D SA Compound 1.0 Tocopheryl Acetate 0.2 Bisabolol QS Parfumol QS Preservative A 3.5 Ceteareth-6, Stearyl Alcohol 1.5 Ceteareth-25 7.5 Ethylhexyl Methoxycinnamate 2.0 Diethylamino Hydroxybenzoyl Hexyl Benzoate 2.0 Cyclopentasiloxane, Cyclohexasiloxane 0.5 Bees Wax 3.0 Cetearyl Alcohol 10.0 Caprylic/Capric Triglyceride B 5.0 Titanium Dioxide, Silica, Methicone, Alumina C 3.0 Glycerin 0.2 Disodium EDTA 0.3 Xanthan Gum 1.0 Decyl Glucoside 2.0 Panthenol, Propylene Glycol 52.3 Aqua dem. D SA Compound 1.0 Tocopheryl Acetate 0.2 Bisabolol QS Perfume QS Preservative Production: Heat Phase A and B separately to approx. 80° C. Add Phase B to Phase A and homogenize by stirring. Heat Phase C to 80° C. and add to the combined Phase A and B and homogenize again. Cool to approx. 40° C. and add Phase D. Homogenize again.

FOOT BALM % Ingredients (INCI) A 2.0 Ceteareth-6, Stearyl Alcohol 2.0 Ceteareth-25 5.0 Cetearyl Ethylhexanoate 4.0 Cetyl Alcohol 4.0 Glyceryl Stearate 5.0 Mineral Oil 0.2 Menthol 0.5 Camphor B 69.3 Aqua dem. QS Preservative C 1.0 Bisabolol 1.0 Tocopheryl Acetate D SA Compound 5.0 Witch Hazel Extract A 2.0 Ceteareth-6, Stearyl Alcohol 2.0 Ceteareth-25 5.0 Cetearyl Ethylhexanoate 4.0 Cetyl Alcohol 4.0 Glyceryl Stearate 5.0 Mineral Oil 0.2 Menthol 0.5 Camphor B 65.3 Aqua dem. QS Preservative C 1.0 Bisabolol 1.0 Tocopheryl Acetate D SA Compound 5.0 Witch Hazel Extract Production: Heat Phase A and B separately to approx. 80° C. Add Phase B to Phase A and homogenize by stirring. Cool to approx. 40° C. and add Phase C and D. Homogenize by stirring and cool to room temperature

W/O % Ingredients (INCI) A 6.0 PEG-7 Hydrogenated Castor Oil 8.0 Cetearyl Ethylhexanoate 5.0 Isopropyl Myristate 15.0 Mineral Oil 0.3 Magnesium Stearate 0.3 Aluminum Stearate 2.0 PEG-45/Dodecyl Glycol Copolymer B 5.0 Glycerin 0.7 Magnesium Sulfate 55.6 Aqua dem. C 1.0 Compound 0.5 Tocopheryl Acetate 0.6 Bisabolol A 6.0 PEG-7 Hydrogenated Castor Oil 8.0 Cetearyl Ethylhexanoate 5.0 Isopropyl Myristate 15.0 Mineral Oil 0.3 Magnesium Stearate 0.3 Aluminum Stearate 2.0 PEG-45/Dodecyl Glycol Copolymer B 5.0 Glycerin 0.7 Magnesium Sulfate 51.6 Aqua dem. C 5.0 Compound 0.5 Tocopheryl Acetate Production: Heat Phase A and B separately to approx. 85° C. Add Phase B to Phase A and homogenize by stirring. Cool to approx. 40° C. and add Phase C. Homogenize by stirring and cool to room temperature.

LIQUID MAKE-UP - TYPE O/W % Ingredients (INCI) A 2.0 Ceteareth-6, Stearyl Alcohol 2.0 Ceteareth-25 6.0 Glyceryl Stearate 1.0 Cetyl Alcohol 8.0 Mineral Oil 7.0 Cetearyl Ethylhexanoate 0.2 Dimethicone B 3.0 Propylene Glycol 1.0 Panthenol QS Preservative 61.9  Aqua dem. C 0.1 Bisabolol SA Compound QS Perfume D 5.7 C.I. 77 891, Titanium Dioxide 1.1 Iron Oxides A 2.0 Ceteareth-6, Stearyl Alcohol 2.0 Ceteareth-25 6.0 Glyceryl Stearate 1.0 Cetyl Alcohol 8.0 Mineral Oil 7.0 Cetearyl Ethylhexanoate 0.2 Dimethicone B 3.0 Propylene Glycol 1.0 Panthenol QS Preservative 57.9  Aqua dem. C 0.1 Bisabolol SA Compound QS Perfume D 5.7 C. I. 77 891, Titanium Dioxide 1.1 Iron Oxides Production: Heat Phase A and B separately to approx. 80° C. Add Phase B to Phase A and homogenize by stirring. Cool to 40° C. and add Phase C and D. Homogenize again and cool to room temperature.

FOUNDATION Ingredient INCI Name % Water Phase Dow Corning 9011 Cyclopentasiloxane, PEG-12 Dimethicone Elastomer Blend Copolymer 15.00 Dow Corning 245 Cyclopentasiloxane 5.00 Fluid Silcare 31 M50 SV Caprylyl Trimethicone 6.35 Propylparaben 0.05 AS 5811 Titanium Dioxide, Triethoxycaprylylsilane 7.50 AS 5131 Iron Oxides, Triethoxycaprylylsilane 0.70 AS 5146 Iron Oxides, Triethoxycaprylylsilane 0.05 AS 5126 Iron Oxides, Triethoxycaprylylsilane 0.35 AS 50230 Talc, Triethoxycaprylylsilane 3.50 Oil Phase Deionized Water 53.30 1.80Butylene Glycol 6.00 Methylparaben 0.20 Benzoic Acid 0.20 Compound SA

The pigments (AS 5811, 5131, 5146, 5126, and 50230; Color Techniques) and propylparaben are dispersed in Silcare 31 M50 SV (Clariant), stirring until wet. The mixture is then passed over a three roll mill at tight setting until particle size is <10 μm. Then, DC 9011 Elastomer Blend (Dow Corning) and DC 245 Fluid are combined in finishing vessel, stirring until homogenous. The color grind is added with slow homogenizer agitation. The water is weighed into a separate vessel and Compound is gradually added with propeller agitation, stirring until dissolved. Methylparaben and benzoic acid are added to butylene glycol. The mixture is warmed slightly, and stirred until dissolved. The mixture is cooled to 30° C. and added to the Compound-containing solution. The water phase is added slowly to the oil phase with rapid agitation. When addition is complete, the preparation is homogenized for five minutes. This preparation is useful as a makeup foundation for application to skin.

Mascara Formulation

The ingredients of both a control preparation and a mascara containing 2% are as follows:

% % Trade Name INCI Name Control 1 Compound 2 Phase # Water Phase  1 Deionized Water 42.96 42.96  2 Butylene Glycol 5.00 5.00  2 Methylparaben 0.30 0.30  3 33-5198 (Black) Iron Oxides 10.00 10.00 (Sun)  4 Natrosol 250 MR Hydroxyethylcellulose 0.20 0.20 (Aqualon)  5 10% KOH Potassium Hydroxide 0.01 0.01  6 Arlacel 165 Glyceryl Stearate, 3.00 3.00 PEG-100 Stearate (Uniqema)  7 10% Citric Acid 0.27 0.27 Phase # Wax Phase  8 Arlacel 165 1.00 1.00  8 Cerasynt SD Glyceryl Stearate (ISP) 3.50 3.50  8 Beeswax, White Beeswax (S&P) 7.50 7.50   SP 424  8 Carnauba #1 Copernica Cerifera 4.80 4.80 (Carnauba) Wax (S&P)  8 Propylparaben 0.10 0.10  9 Deionized Water 20.00 —  9 10% Compound/ — 20.00   Water 10 Deionized Water 1.00 1.00 10 Glydant DMDM Hydantoin 0.36 0.36 (Lonza) 100.00 100.00

To produce the mascara formulation, the wax phase 8 is combined and heated to 85-90° C. with propeller mixing. The 10% Compound solution is prepared by adding Compound to water while propeller mixing. Phase 1 water is added to a tared stainless steel beaker (approximately 50 g excess is added to compensate for loss). Phase 2 methylparaben is added to butylene glycol and stirred while warming on top of a steam bath until dissolved, then added to the water with slow homomixer agitation. Then, the phase 4 black iron oxide is added, while maintaining agitation. Then, Natrosol is sprinkled in, while maintaining agitation. The 10% KOH is added, and heating is begun to 85° C., with the beaker covered as tightly as possible. When the Natrosol is dissolved, the 10% citric acid is added dropwise, maintaining temperature and agitation. Then, the Arlacel 165 is added slowly and mixed for at least 5 minutes to insure dissolution. At 85-90° C., the wax phase is slowly added to the water phase while homomixing. The temperature and agitation are maintained for 10 minutes. The batch is removed from the steam bath and allowed to cool while homomixing with occasional hand scraping of the beaker walls. At 55° C., the batch is weighed to check for water loss. Mixing is resumed and water is added back, if necessary. At 45° C., phases 9 and 10 are added. Cooling is continued using cold water to 30° C. At this point, continuous hand scraping of beaker walls is necessary.

In this preparation, the small amount of KOH (in Phase 5) is used to raise the pH to disperse the Natrosol which is coated with glyoxal to retard wetting, and prevent agglomeration. In phase 7, the citric acid is added slowly to adjust pH to 5.5, below the isoelectric point of the iron oxides. In phases 7 and 8, the Arlacel 165 is split between the oil and water phases, as the emulsification is easier to accomplish with surfactant in both phases. In phase 9, the deionized water is added in the control batch instead of Compound. The Compound solution is prepared while the emulsion is being processed, so it is absolutely fresh. This preparation provides a formulation suitable for use as a mascara.

Example 18 Hair Growth Inhibition

In this Example, experiments to determine the ability of the compositions of the present invention to inhibit the growth of hair are described. In particular, these experiments are conducted in order to assess the ability of the compositions of the present invention to decrease hair growth after depilation by shaving or use of depilatory creams or waxing.

A lotion for inhibiting hair growth and containing a modified variant BBPI in which the chymotrypsin loop of the parent BBPI is replaced with a VEGF-binding peptide is prepared according to the following formulation (A):

Ingredient (INCI name) % in weight Water 85.97 Cetearyl alcohol 8.50 Ceteareth-20 2.70 PPG-15 Stearyl Ether 0.60 Prunus Amygdalus Dulcis Oil (1) 0.10 Paraffinum Liquidum 0.10 Allantoin 0.20 Propylene glycol 0.13 CI77891 Titanium dioxide 0.17 Tetra sodium EDTA 0.10, VEGF-BBPI 0.10 Sodium Chloride 0.091 Citric acid 0.11 Sodium Hydroxyde 0.034 Phenoxyethanol 1.0 Robertet Artlande G10029876 (2) 0.10

The composition includes at least one type of VEGF-BBPI chosen from SEQ ID NOS: 601, 602, 627-631, 643, 491, 632-636. In some embodiments, the composition comprises at least two, at least three, at least four or at least five different types of VEGF-BBPIs chosen from SEQ ID NOS:601, 602, 627-631, 643, 491, 632-636.

The formulation comprising the VEGF-BBPI is manufactured as follows:

1) Blending the fatty alcohol emulsifier and oil gelling agent together into a molten phase at a temperature of 60, preferably 70° C. or more,

2) emulsifying the molten phase into an aqueous phase, the temperature of the aqueous phase prior to emulsification being 50° C., preferably 60° C., more preferably 70° C. or more, whereby an emulsion is formed,

3) cooling the emulsion to a temperature of 35° C. or less,

4) dispersing the perfume, preservative, citric acid solution buffer solution in the emulsion.

5) adding in the same manner the solution of VEGF-BBPI, finishing with the buffer solution over a period of approximately 5 min.

6) Agitating the mixture for a further 10 minutes

A control formulation (B) is prepared according to the method described for the preparation of formulation A but excluding the VEGF-BBPI.

Facial Hair Experiments

In these experiments, a group (e.g., 5) male subjects with Fitzpatrick Skin Classification II are tested. Individuals are requested to use no topical facial treatment prior to beginning the experiments. On day 1, facial hair growth is visually evaluated and photographed. Following this evaluation and photography, the composition(s) to be tested, as well as a vehicle control are applied at the desired concentration(s). Beginning on day 2, the individuals apply the composition(s) immediately after shaving. No other pre- or post-shave treatment is used for the duration of the experiments. In most cases, the experiment continues for a time period of 30 to 45 days. Facial hair growth is visually evaluated and photographed every third day during the experiments. The number of hairs, as well as the hair shaft length and width are measured using computerized image analysis. In preferred embodiments, there is a decrease in the number of hairs, hair thickness and/or hair length due to the application of the test compound(s).

Leg Hair Experiments

In these experiments, a group (e.g., 5) female subjects with Fitzpatrick Skin Classification II are tested. Individuals are requested to use no topical leg treatment prior to beginning the experiments. On day 1, areas on both legs of each individual are marked and the hair growth is visually evaluated and photographed. Following this evaluation and photography, the composition(s) to be tested are applied at the desired concentration(s). Following this evaluation and photography, the composition(s) to be tested (i.e., test compounds containing a desired concentration of VEGF-BBP), as well as a vehicle control, are applied at the desired concentration(s). In some methods, each individual is provided with two tubes, one of which contains the VEGF-BBPI and the other containing the vehicle control. These tubes are marked “left” and “right.” Each day during the experiments, the subject applies the compositions in the two tubes the respective legs. After 7 days of application, the individuals are visually evaluated and photographs are taken. Both legs are then shaved or exposed to a depilatory and the test individuals continue to apply the compositions as before. Hair growth is then evaluated visually and by photographing appropriate areas on the legs every 2 days. After 10 days, the legs are again shaved and the test subjects continue to apply the compositions as before. In some methods, the experiments are conducted for 3 cycles and the hair growth is visually evaluated and photographs were taken. The experiments are then continued for an additional 8 days. In preferred embodiments, there is a decrease in the number of hairs, hair thickness and/or hair length due to the application of the test compound(s) in the marked area(s).

Beginning on day 2, the individuals apply the composition(s) immediately after shaving. No other pre- or post-shave treatment is used for the duration of the experiments. In most cases, the experiment continues for a time period of 30 to 45 days. Facial hair growth is visually evaluated and photographed every third day during the experiments. The number of hairs, as well as the hair shaft length and width are measured using computerized image analysis. In preferred embodiments, there is a decrease in the number of hairs, hair thickness and/or hair length due to the application of the test compound(s). 

1. An isolated modified variant Bowman Birk Protease Inhibitor (BBPI), wherein the second protease inhibitory loop of the BBPI scaffold is a binding peptide, and wherein said modified variant BBPI further comprises a substituted amino acid at least at one amino acid position chosen from positions equivalent to 1, 4, 5, 11, 13, 18, 25, 27, 29, 31, 38, 40, 50, 52, 55, and 65 of the variant BBPI of SEQ ID NO:187.
 2. The isolated modified variant BBPI of claim 1, wherein said BBPI scaffold is chosen from BBI (SEQ ID NO:13), BBIt (SEQ ID NO:185), BBI-AV (SEQ ID NO:186), BBIt-AV (SEQ ID NO:187), BBIt-VEGK (SEQ ID NO:640), BBIt-VEGT (SEQ ID NO:641), BBIt-VEGKD (SEQ ID NO:642), BBdb (SEQ ID NO:449), BBsb3 (SEQ ID NO:450), BBtc (SEQ ID NO:451), BBdb-AV (SEQ ID NO:452), BBsb3-AV (SEQ ID NO:453) and BBtc-AV (SEQ ID NO:454).
 3. The isolated modified variant BBPI of claim 1, wherein said binding peptide is chosen from a VEGF binding peptide, an FGF-5 binding peptide, a TGFβ binding peptide and a TNFα binding peptide.
 4. The isolated modified variant BBPI of claim 1, wherein said binding peptide is a VEGF binding peptide chosen from ACYNLYGWTC (SEQ ID NO:9), KYYLYWW (SEQ ID NO:458), TLWKSYW (SEQ ID NO:459), DLYWW (SEQ ID NO:460), SKHSQIT (SEQ ID NO:468) KTNPSGS (SEQ ID NO:469) RPTGHSL (SEQ ID NO:470), KHSAKAE (SEQ ID NO:471) KPSSASS (SEQ ID NO:472), PVTKRVH (SEQ ID NO:473), TLHWWVT (SEQ ID NO:492), PYKASFY (SEQ ID NO:493), PLRTSHT (SEQ ID NO:494), EATPROT (SEQ ID NO:495), NPLHTLS (SEQ ID NO:496), KHERIWS (SEQ ID NO:497), ATNPPPM (SEQ ID NO:498), STTSPNM (SEQ ID NO:499), ADRSFRY (SEQ ID NO:500), PKADSKQ (SEQ ID NO:501), PNQSHLH (SEQ ID NO:502), SGSETWM (SEQ ID NO:503), ALSAPYS (SEQ ID NO:504), KMPTSKV (SEQ ID NO:505), ITPKRPY (SEQ ID NO:506), KWIVSET (SEQ ID NO:507), PNANAPS (SEQ ID NO:508), NVQSLPL (SEQ ID NO:509), TLWPTFW (SEQ ID NO:510), NLWPHFW (SEQ ID NO:511), SLWPAFW (SEQ ID NO:512), SLWPHFW (SEQ ID NO:513), APWNSHI (SEQ ID NO:514), APWNLHI (SEQ ID NO:515), LPSWHLR (SEQ ID NO:516), PTILEWY (SEQ ID NO:517), TLYPQFW (SEQ ID NO:518), HLAPSAV (SEQ ID NO:519), KYYLSWW (SEQ ID NO:520), WYTLYKW (SEQ ID NO:521), TYRLYWW (SEQ ID NO:522), RYSLYYW (SEQ ID NO:523), YYLYYWK (SEQ ID NO:524), NYQLYGW (SEQ ID NO:525), TKWPSYW (SEQ ID NO:226), TLWKSYW (SEQ ID NO:527), PLWPSYW (SEQ ID NO:528), RLWPSYW (SEQ ID NO:529), TLWPKYW (SEQ ID NO:530), KYDLYWW (SEQ ID NO;531), RYDLYWW (SEQ ID NO:532), DYRLYWW (SEQ ID NO:533), DYKLYWW (SEQ ID NO:534), EYKLYWW (SEQ ID NO:535), and RYPLYWW (SEQ ID NO:536).
 5. The isolated modified variant BBPI of claim 1, wherein said binding peptide is an FGF-5 binding peptide chosen from CACRTQPYPLCF (MM007; SEQ ID NO:430), CICTWIDSTPC (PS2; SEQ ID NO:431), CYGLPFTRC (SEQ ID NO:537), CEEIWTMLC (SEQ ID NO:538), CWALTVKTC (SEQ ID NO:539), CLTVLWTTC (SEQ ID NO:540), CTLWNRSPC (SEQ ID NO:541), CHYLLTNYC (SEQ ID NO:542), CRIHLAHKC (SEQ ID NO:543), TNIDSTP (SEQ ID NO:544), HLQTTET (SEQ ID NO:545), SLNNLTV (SEQ ID NO:546), TNIDSTP (SEQ ID NO:547), TNIDSTP (SEQ ID NO:548), LRILANK (SEQ ID NO:549), LLTPTLN (SEQ ID NO:550), ALPTHSN (SEQ ID NO:551), TNIDSTP (SEQ ID NO:552), LCRRFEN (SEQ ID NO:553), TNIDSTP (SEQ ID NO:554), TNIDSTP (SEQ ID NO:555), HLQTTET (SEQ ID NO:556), PLGLCPP (SEQ ID NO:557), GYFIPSI (SEQ ID NO:558), TKIDSTP (SEQ ID NO:559), HLQTTET (SEQ ID NO:560), WNIDSTP (SEQ ID NO:561), TWIDWTP (SEQ ID NO:562), RTQPYPL (SEQ ID NO:670) and TWIDSTP (SEQ ID NO:671).
 6. The isolated modified variant BBPI of claim 1, wherein said binding peptide is a TGFβ binding peptide chosen from CLCPENINVLPCN (PEN3; SEQ ID NO:436), CICKHNVDWLCF (MMO21W; SEQ ID NO:437), CICWTQHIHNCF (WTQ; SEQ ID NO:438), CVTTDWIEC (SEQ ID NO:563), CYYSQFHQC (SEQ ID NO:564), CPTLWTHMC (SEQ ID NO:565), QSACIVYYVGRKPKVECASSD (SEQ ID NO:566), QSACILYYIGKTPKIECASSD (SEQ ID NO:567), QSACILYYVGRTPKVECASSD (SEQ ID NO:568), acetyl-LCPENDNVSPCY-cohn2 (SEQ ID NO:569), KHNVRLL (SEQ ID NO:570), NDTPSYF (SEQ ID NO:571), AKLYAGS (SEQ ID NO:572), RGPAHSL (SEQ ID NO:573), NSLAERR (SEQ ID NO:574), HPLASPH (SEQ ID NO:575), QPWNKLK (SEQ ID NO:576), AWLr/Mipy (SEQ ID NO:577), PTKPAQQ (SEQ ID NO:578), PSLNRPQ (SEQ ID NO:579), HHARQEW (SEQ ID NO:580), RHHTPGP (SEQ ID NO:581), ASAINPH (SEQ ID NO:582), CHGYDRAPC (SEQ ID NO:644), CFAPADQAC (SEQ ID NO:645), CIPSRFITC (SEQ ID NO:646), CHGHTKLAC (SEQ ID NO:647), CNGKSKLAC (SEQ ID NO:648), PENINVLP (SEQ ID NO;672), KHNVDWL (SEQ ID NO:673) and WTQHIHNC (SEQ ID NO:674).
 7. The isolated modified variant BBPI of claim 1, wherein said binding peptide is a TNFα binding peptide chosen from RYWQDIP (T1; SEQ ID NO:474), APEPILA (T2; SEQ ID NO:475), DMIMVSI (T3; SEQ ID NO:476), WTPKPTQ (SEQ ID NO:583), ATFPNQS (SEQ ID NO:584), ASTVGGL (SEQ ID NO:585), TMLPYRP (SEQ ID NO:586), AWHSPSV (SEQ ID NO:587), TQSFSS (SEQ ID NO:588), THKNTLR (SEQ ID NO:589), GQTHFHV (SEQ ID NO:590), LPILTQT (SEQ ID NO:591), SILPVSH (SEQ ID NO:592), SQPIPI (SEQ ID NO:593), and QPLRKLP (SEQ ID NO:594).
 8. The isolated modified variant BBPI of claim 1, wherein said substituted amino acid at position 1 is chosen from A and C.
 9. The isolated modified variant BBPI of claim 1, wherein said at least one substitution at position 4 is V.
 10. The isolated modified variant BBPI of claim 1, wherein said at least one substitution at position 5 is chosen from P, and A.
 11. The isolated modified variant BBPI of claim 1, wherein said at least one substitution at position 11 is 11G.
 12. The isolated modified variant BBPI of claim 1, wherein said at least one substitution at position 13 is chosen from 13Y, 13I, 13F, 13M, 13L, 13V, 13K, and 13R.
 13. The isolated modified variant BBPI of claim 1, wherein said at least one substitution at position 18 is chosen from 18I and 18V and 18L.
 14. The isolated modified variant BBPI of claim 1, wherein said at least one substitution at position 25 is chosen from 25K, 25N, 25W, 25I, 25A and 25R.
 15. The isolated modified variant BBPI of claim 1, wherein said at least one substitution at position 27 is chosen from 27H, 27K, 27V, 27A, and 27Q.
 16. The isolated modified variant BBPI of claim 1, wherein said at least one substitution at position 29 is chosen from 29R, 29K, and 29P.
 17. The isolated modified variant BBPI of claim 1, wherein said at least one substitution at position 31 is chosen from 31Q, 31H, 31E, 31A, 31R, 31W, 31K and 31T.
 18. The isolated modified variant BBPI of claim 1, wherein said at least one substitution at position 38 is chosen from 38N, 38K, and 38R.
 19. The isolated modified BBI of claim 1, wherein said at least one substitution at position 40 is chosen from 40H, 40K, 40Q, 40R, and 40Y.
 20. The isolated modified variant BBPI of claim 1, wherein said at least one substitution at position 50 is chosen from 50R, 50Q, 50K, 50T, 50V, 50M, and 50S.
 21. The isolated modified variant BBPI of claim 1, wherein said at least one substitution at position 52 is chosen from 52K, 52T, 52R, 52Q, 52L, 52H, 52A, 52M, 52S and 52E.
 22. The isolated modified variant BBPI of claim 1, wherein said at least one substitution at position 55 is 55M.
 23. The isolated modified variant BBPI of claim 1, wherein said at least one substitution at position 65 is chosen from 65E, 65Q, and 65D.
 24. The isolated modified variant BBPI of claim 1, further comprising an insert of SEQ ID NO:
 389. 25. The isolated modified variant BBPI of claim 1, wherein said modified variant BBPI is expressed as a fusion protein comprising the catalytic domain chosen from cellulase, cutinase and disulfide isomerase.
 26. The isolated modified variant BBPI of claim 1, wherein said modified variant BBPI is expressed as the fusion protein of SEQ ID NO:195.
 27. The isolated modified variant BBPI of claim 1, wherein said modified variant BBPI comprises a combination of two amino acid substitutions at amino acid positions equivalent to positions 50 and 52 of SEQ ID NO:187.
 28. The isolated modified variant BBPI of claim 27, wherein said modified variant BBPI has the amino acid sequence of SEQ ID NO:595.
 29. The isolated modified variant BBPI of claim 1, wherein said modified variant BBPI comprises a combination of three amino acid substitutions chosen from amino acid substitutions at positions equivalent to positions 25, 29, 40, 50 and 52 of SEQ ID NO:187.
 30. The isolated modified variant BBPI of claim 29, wherein said combination of three amino acids is chosen from combinations 25L-50T-52A, 29P-50T-52A, 40K-50T-52A.
 31. The isolated modified variant BBPI of claim 29, wherein said modified variant BBPI an amino acid sequence chosen from SEQ ID NOS:603, 607 and
 609. 32. The isolated modified variant BBPI of claim 1, wherein said modified variant BBPI comprises a combination of four amino acid substitutions chosen from amino acid substitutions at positions equivalent to positions 13, 29, 50 and 52 of SEQ ID NO:187.
 33. The isolated modified BBPI of claim 32, wherein said combination of four amino acids is chosen from combinations 13I-25L-50T-52A, 13I-29P-50T-52A, 13I-A0K-50T-52A, 25L-29P-50T-52A, 25L-40K-50T-52A, and 29P-40K-50T-52A.
 34. The isolated modified variant BBPI of claim 33, wherein said modified variant BBPI an amino acid sequence chosen from SEQ ID NOS:596, 600, 602, 604, 606, 608,and
 643. 35. The isolated modified variant BBPI of claim 1, wherein said modified variant BBPI comprises a combination of five amino acid substitutions chosen from amino acid substitutions at positions equivalent to positions 13, 25, 29, 40, 50, and 52 of SEQ ID NO:187.
 36. The isolated modified variant BBPI of claim 35, wherein said combination of five amino acids is chosen from combinations 13I-25L-29P-50T-52A, 13I-25L-40K-50T-52A, A13I-29P-40K-50T-52A, 25L-29P-40K-50T-52A, 13I-29P-40K-50K-52A, and 13I-29P-40K-50T-52T.
 37. The isolated modified variant BBPI of claim 36, wherein said modified variant BBPI an amino acid sequence chosen from SEQ ID NOS:432, 434, 443, 445, 446, 597, 599, 601, 605, 615, 620, 624, and
 625. 38. The isolated modified variant BBPI of claim 1, wherein said modified variant BBPI comprises a combination of six amino acid substitutions chosen from amino acid substitutions at positions equivalent to positions 1, 4, 5, 11, 13, 25, 29, 40, 50 and 52 of SEQ ID NO:187.
 39. The isolated modified variant BBPI of claim 38, wherein said combination of six amino acids is chosen from combinations 13I-25L-29P-40K-50T-52A, 1C-13I-29P-40K-50T-52A, 4V-13I-29P-40K-50T-52A, 5P-13I-29P-40K-50T-52A, 11G-13I-29P-40K-50T-52A, 13I-25R-29P-40K-50T-52A, 13I-27R-29P-40K-50T-52A, 13I-29P-31A-40K-50T-52A, 13I-29P-31R-40K-50T-52A, 13I-29P-38N-40K-50T-52A, and 13I-29P-40K-50T-52A-65E.
 40. The isolated modified variant BBPI of claim 39, wherein said modified variant BBPI an amino acid sequence chosen from SEQ ID NOS:598, 611, 612, 613, 614, 616, 619, 621, 622, 623, and
 626. 41. The isolated modified variant BBPI of claim 1, wherein said modified variant BBPI comprises a combination of seven amino acid substitutions chosen from amino acid substitutions at positions equivalent to positions 13, 25, 29, 31, 40, 50 and 52 of SEQ ID NO:187.
 42. The isolated modified variant BBPI of claim 41, wherein said combination of seven amino acids is chosen from combinations 13L-25R-29P-31A-40K-50T-52A, 13L-25R-29P-31R-40K-50T-52A and 13I-25R-27A-29P-31A-50K-52T.
 43. The isolated modified variant BBPI of claim 41, wherein said modified variant BBPI an amino acid sequence chosen from SEQ ID NOS:491, 617, 618, and 632-639.
 44. The isolated modified variant BBPI of claim 1, wherein said modified variant BBPI comprises a combination of eight amino acid substitutions chosen from amino acid substitutions at positions equivalent to positions 13, 25, 27, 29, 31, 40, 50 and 52 of SEQ ID NO:187.
 45. The isolated modified BBPI of claim 44, wherein said combination of eight amino acids is chosen from combinations 13I-25R-27A-29P-31A-40H-50K-52T, 13I-25K-27A-29R-31E-40K-50Q-52Q, 13I-25K-27R-29E-31A-40H-50R-52K, 13I-25K-27A-29R-31A-40H-50R-52L and 13I-25K-27Q-29P-31E-40H-50R-52Q.
 46. The isolated modified variant BBPI of claim 44, wherein said modified variant BBPI an amino acid sequence chosen from SEQ ID NOS:627-631.
 47. The isolated modified variant BBPI of claim 1, wherein said modified variant BBPI binds VEGF.
 48. The isolated modified variant BBPI of claim 1, wherein said VEGF binding peptide is chosen from SEQ ID NOS:9, 458, 459, 460, 468, 469, 470, 471, 472 and
 473. 49. The isolated modified variant BBPI of claim 1, wherein said modified variant BBPI is a VEGF-BBPI chosen from SEQ ID NOS:601, 602, 627, 628, 630, 631, 643, 491, 632, 633, 634, 635, and
 636. 50. The isolated modified variant BBPI of claim 1, wherein said isolated modified variant inhibitor, has greater trypsin inhibitory activity than the corresponding unmodified precursor variant BBPI.
 51. The isolated modified variant BBPI of claim 1, wherein said isolated modified variant BBPI, has greater trypsin inhibitory activity and production yield than the corresponding unmodified precursor variant BBPI.
 52. An isolated modified variant BBPI of SEQ ID NO:187, wherein said BBPI comprises an amino acid substitution at least at one amino acid position chosen from amino acid substitutions at positions equivalent to positions 13, 25, 27, 29, 31, 40, 50 and 52, and wherein the VEGF variant peptide of said BBPI is replaced by a variant peptide to bind a target protein chosen from FGF-5, TGFβ and TNFα.
 53. The isolated modified variant BBPI of claim 52, wherein said variant peptide is an FGF5 binding peptide chosen from SEQ ID NOS: 430, 431, 670, and
 671. 54. The isolated modified variant BBPI of claim 52, wherein said binding peptide is a TGFβ binding peptide chosen from SEQ ID NOS:436, 437, 438, 672, 673, and
 674. 55. The isolated modified variant BBPI of claim 52, wherein said binding peptide is a TNFα binding peptide chosen from SEQ ID NOS:474, 475, and
 476. 56. The isolated modified variant BBPI of claim 52, wherein said modified variant BBPI has greater trypsin inhibitory activity and production yield than the corresponding unmodified precursor BBPI.
 57. The isolated modified variant BBPI of claim 52, wherein said variant Bowman Birk Inhibitor is chosen from SEQ ID NO:432, 434, 443, 445, 447, 637, 638, and
 639. 58. An isolated polynucleotide encoding a BBPI fusion protein, said polynucleotide comprising a first polynucleotide sequence encoding the catalytic unit of an enzyme, and a second polynucleotide sequence encoding a modified variant protease inhibitor chosen from a VEGF-binding BBPI (VEGF-BBPI), an FGF-binding BBPI (FGF-BBPI), a TGF-binding BBPI (TGF-BBPI) and a TNF-binding BBPI (TNF-BBPI).
 59. The isolated polynucleotide of claim 58, wherein said first sequence encodes the catalytic unit of a cellulase.
 60. A method for producing a modified variant BBPI in a bacterial cell, said method comprising: a) mutating a polynucleotide that encodes a variant BBPI to generate a polynucleotide construct encoding a modified variant BBPI comprising an amino acid substitution at least at one amino acid position chosen from positions equivalent to 1, 4, 5, 11, 13, 18, 25, 27, 29, 31, 38, 40, 50, 52, 55, and 65 of the variant BBPI of SEQ ID NO:187; b) introducing said polynucleotide construct into a bacterial host cell; c) culturing said bacterial cell under suitable culture conditions to allow expression of said heterologous DNA sequence; and d) producing said modified variant BBPI, wherein said modified variant BBPI has a greater trypsin inhibitory activity than the corresponding unmodified precursor variant BBPI.
 61. The method of claim 60, wherein said modified variant BBPI has a greater trypsin inhibitory activity and production yield than the corresponding unmodified precursor variant BBPI.
 62. The method of claim 60, wherein said unmodified precursor variant BBPI is chosen from BBI-AV (SEQ ID NO:186), BBIt-AV (SEQ ID NO:187), BBdb-AV (SEQ ID NO:452), BBsb3 (SEQ ID NO:453), and BBtc-AV (SEQ ID NO:454),
 63. The method of claim 60, wherein the second protease inhibitory loop of said modified variant BBPI is a target protein binding peptide chosen from a VEGF binding peptide, an FGF-5 binding peptide, a TGFβ binding peptide and a TNFα binding peptide.
 64. The method of claim 60, further comprising recovering said modified variant BBPI.
 65. The method of claim 60, further comprising activating said modified variant BBPI.
 66. The method of claim 60, wherein said modified variant BBPI comprises a combination of amino acid substitutions chosen from combinations (3) 25L-50T-52A, 29P-50T-52A, 40K-50T-52A, (4) 13I-25L-50T-52A, 13I-29P-50T-52A, 13I-A0K-50T-52A, 25L-29P-50T-52A, 25L-40K-50T-52A, and 29P-40K-50T-52A, (5) 13I-25L-29P-50T-52A, 13I-25L-40K-50T-52A, 25L-29P-40K-50T-52A, 13L-29P-40K-50T-52A, 13I-29P-40K-50K-52A, and 13L-29P-40K-50T-52T, (6) 13I-25L-29P-40K-50T-52A, 1C-13I-29P-40K-50T-52A, 4V-13I-29P-40K-50T-52A, 5P-13I-29P-40K-50T-52A, 11G-13I-29P-40K-50T-52A, 13I-25R-29P-40K-50T-52A, 13I-27R-29P-40K-50T-52A, 13I-29P-31A-40K-50T-52A, 13I-29P-31R-40K-50T-52A, 13I-29P-38N-40K-50T-52A, 13I-29P-40K-50T-52A-65E, (7) 13L-25R-29P-31A-40K-50T-52A, and 13L-25R-29P-31R-40K-50T-52A, (8) 13I-25R-27A-29P-31A-40H-50K-52T, 13I-25K-27A-29R-31E-40K-50Q-52Q, 13I-25K-27R-29E-31A-40H-50R-52K, 13I-25K-27A-29R-31A-40H-50R-52L, 13I-25K-27Q-29P-31E-40H-50R-52Q.
 67. An expression vector comprising a polynucleotide sequence that encodes the modified variant BBPI of claim
 1. 68. The expression vector of claim 67, wherein said modified variant BBPI is expressed as a fusion protein having at least 80% identity to the polypeptide of SEQ ID NO:195.
 69. The expression vector of claim 67, wherein the second protease inhibitory loop of said modified BBPI is a binding peptide chosen from a VEGF binding peptide, an FGF5 binding peptide, a TGFβ binding peptide and a TNFα binding peptide.
 70. A host cell transformed with the vector of claim
 67. 71. The host cell of claim 70, wherein said host cell is a Bacillus species host cell. 